Peptide nucleic acid conjugates

ABSTRACT

The present disclosure is directed to conjugates of a specific binding entity and an oligomer, i.e. [Specific Binding Entity]-[Oligomer] n , wherein n is an integer ranging from 1 to 12, and where the Oligomer includes, in some embodiments, a PNA sequence having at least one substituent at a gamma carbon position. In some embodiments, the substituent at the gamma carbon position, e.g. an amino acid, a peptide, a miniPEG, or a polymer, includes at least one reporter moiety.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/EP2018/065697 filed on Jun. 13, 2018, which application claimsthe benefit of the filing date of International Application No.PCT/US2017/66976 filed on Dec. 18, 2017; and which also claims thebenefit of the filing date of U.S. Provisional Patent Application No.62/599,810 filed Dec. 18, 2017, the disclosures of which are herebyincorporated herein by reference in their entireties.

BACKGROUND OF THE DISCLOSURE

Cell staining methods, including immunohistochemistry (IHC) and in situhybridization analysis (ISH), are useful tools in histological diagnosisand the study of tissue morphology. IHC employs specific binding agentsor moieties, such as antibodies, to detect an antigen of interest thatmay be present in a tissue sample. IHC is widely used in clinical anddiagnostic applications, such as to diagnose particular disease statesor conditions. For example, particular cancer types can be diagnosedbased on the presence of a particular marker molecule in a sampleobtained from a subject. IHC is also widely used in basic research tounderstand biomarker distribution and localization in different tissues.Biological samples also can be examined using in situ hybridizationtechniques, such as silver in situ hybridization (SISH), chromogenic insitu hybridization (CISH) and fluorescence in situ hybridization (FISH),collectively referred to as ISH. ISH is distinct from IHC in that ISHdetects nucleic acids in tissue whereas IHC detects proteins in tissue.

Characterization and quantitation of the multitude of proteins expressedby an organism's genome are the focus of proteomics. Multipleximmunohistochemistry (mIHC) represents a major unmet technological needto detect and analyze multivariate protein targets in paraffin-embeddedformalin-fixed tissues with broad applications in research anddiagnostics. Multiplex immunohistochemistry (mIHC) techniques areattempting to address the need for detecting and analyzing multivariateprotein targets in formalin-fixed, paraffin-embedded (FFPE) tissues.Effective mIHC techniques have broad applications in research anddiagnostics. However, there are few, if any, efficient and reproduciblemethods that allow simultaneous and quantitative detection of multipleprotein targets in tissues.

A key constraint of translational research within a clinical trialsetting is that there is often a limited amount of tissue from which tocarry out biomarker analyses. Further, this tissue is frequentlyarchived and stored in FFPE blocks. Traditional methods of geneexpression analysis have limitations for clinical application. Forexample, RT-PCR measures the expression of one gene at a time, whereasmultiplex expression profiling techniques such as microarrays, coveringmany thousands of transcripts, are often expensive and lack flexibilityand reproducibility when evaluating low-quality RNA samples such asthose from FFPE. The evaluation of these assays is semi-quantitative andinherently subjective. This has led to an increase focus on developingquantitative and highly multiplexed assays that enable profiling ofmultiple markers with a single assay. Platforms that enable multiplexedanalysis of biomarkers from limited amounts of poor-quality material aretherefore very attractive.

NanoString nCounter technology, which enables direct automated detectionof nucleic acids (DNA and/or RNA) is a relatively new technology thathas been employed in various clinical and research applications.Commonly, the target nucleic acid (DNA or RNA) is hybridized to abiotinylated DNA strand (capture strand,) enabling the immobilization ofthe nucleic acid to a streptavidin surface inside the nCounter cartridgeand a fluorescently labeled DNA strand (reporter strand, 7 Kb of whichabout 50 bases are complementary to the target nucleic acid). Theautomated digital readout of the fluorescently labeled reporter strandis believed to allow for the non-amplified measurement of up to 800nucleic acid targets within one sample.

DNA antibody barcoding consists of labeling an antibody with a cleavableDNA strand that can be used as a unique molecular tag. Combining DNAantibody barcoding with the NanoString nCounter technology expanded theNanoString nCounter technology to encompass applications involvingmultiplexed protein analysis.

Synthetic molecules that can bind with high affinity and sequencespecificity to a chosen target in a gene sequence are of interest inmedicinal and biotechnological contexts. They show promise for thedevelopment of gene therapeutic agents, diagnostic devices for geneticanalysis, and as molecular tools for nucleic acid manipulations. Peptidenucleic acid (PNA) is a nucleic acid analog in which the sugar phosphatebackbone of natural nucleic acid has been replaced by a syntheticpeptide backbone usually formed from N-(2-amino-ethyl)-glycine units,resulting in an achiral and uncharged mimic. It is believed to bechemically stable and resistant to hydrolytic (enzymatic) cleavage andthus not expected to be degraded inside a living cell. PNA is capable ofsequence-specific recognition of DNA and RNA obeying the Watson-Crickhydrogen bonding scheme, and the hybrid complexes exhibit extraordinarythermal stability and unique ionic strength effects. Since PNA containsno charges, the binding hybridization between PNA and DNA is strongerthan that between DNA and DNA for the same sequence.

BRIEF SUMMARY OF THE DISCLOSURE

In one aspect of the present disclosure is a conjugate having thestructure of Formula (IA):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody (e.g. a primary antibody or a secondary antibody), an antibodyfragment, a drug/antibody complex, and a nucleic acid;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

Z is selected from the group consisting of a PNA sequence including atleast one nucleotide having a substituent at a gamma carbon position, anuncharged DNA sequence, and a DNA sequence comprising charged anduncharged bases;

X is a label;

Y is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms;

m is 0 or an integer ranging from 1 to 6;

z is 0 or 1; and

n is an integer ranging from 1 to 12.

In some embodiments, X is biotin, an enzyme, a chromogen, a fluorophore,a hapten, or a mass spectrometry tag.

In some embodiments, the ‘Specific Binding Entity’ is a primaryantibody. In some embodiments, the ‘Specific Binding Entity’ is asecondary antibody. In some embodiments, the ‘Specific Binding Entity’is a nucleic acid (e.g. DNA or RNA).

In some embodiments, Z includes a DNA sequence comprising only unchargedDNA bases. In some embodiments, Z comprises a DNA sequence comprising amixture of charged and uncharged bases. In some embodiments, Z comprisesa DNA sequence in which at least 50% of the bases in the DNA sequenceare uncharged.

In some embodiments, Z comprises a PNA sequence having at least two PNAbases having substituents at a gamma position. In some embodiments, Zcomprises a PNA sequence having at least three PNA bases havingsubstituents at a gamma position. In some embodiments, Z comprises a PNAsequence having at least four PNA bases having substituents at a gammaposition.

In some embodiments, the at least one substituted nucleotide of the PNAsequence includes a lysine residue or a lysine residue including areporter moiety. In some embodiments, the at least one substitutednucleotide of the PNA sequence includes a miniPEG including at least onereporter moiety. In some embodiments, the at least one substitutednucleotide of the PNA sequence includes a polymer having at least onereporter moiety. In some embodiments, the at least one substitutednucleotide of the PNA sequence includes a peptide including at least onereporter moiety (e.g. a chromogen, a fluorophore).

In some embodiments, the PNA sequence comprises between about 5 and 20bases. In some embodiments, the PNA sequence comprises between about 5and 15 bases. In some embodiments, the PNA sequence comprises betweenabout 5 and 10 bases. In some embodiments, the PNA sequence comprisesabout 15 bases. In some embodiments, the PNA sequence comprises about 10bases.

In some embodiments, the ‘Specific Binding Entity’ is a primary antibodyand Z is a PNA sequence or PNA sequence having one or more substituentsat a gamma position. In some embodiments, the ‘Specific Binding Entity’is a primary antibody and Z is a PNA sequence having 10 nucleotides. Insome embodiments, the ‘Specific Binding Entity’ is a primary antibodyand Z is a PNA sequence having one or more substituents at a gammaposition.

In some embodiments, the ‘Linker’ comprises a group which is capable ofbeing cleaved, e.g. a photocleavable group, an enzymatically cleavagegroup, a chemically cleavable group, a group cleavable at certain pHs.In some embodiments, the ‘Linker’ comprises one or more groups whichimpart water solubility as disclosed herein (e.g. one or more PEGgroups).

In another aspect of the present disclosure is a conjugate having thestructure of Formula (IIB):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody, an antibody fragment, a drug/antibody complex, and a nucleicacid;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

‘PNA’ is a PNA sequence including at least one nucleotide having asubstituent at a gamma carbon position;

X is a label;

Y is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms;

m is 0 or an integer ranging from 1 to 6;

z is 0 or 1; and

n is an integer ranging from 1 to 12.

In some embodiments, X is biotin, an enzyme, a chromogen, a fluorophore,a hapten, and a mass spectrometry tag.

In some embodiments, the PNA sequence has at least two gamma PNA bases.In some embodiments, each of the at least two gamma PNA bases issubstituted with the same amino acid, miniPEG, peptide, or polymer. Insome embodiments, each of the at least two gamma PNA bases issubstituted with a different amino acid, miniPEG, peptide, or polymer.In some embodiments, the amino acid, miniPEG, peptide, or polymerincludes at least one reporter moiety (e.g. a fluorophore, a chromogen).In some embodiments, the PNA sequence has at least three gamma PNAbases. In some embodiments, the PNA sequence has at least four gamma PNAbases.

In some embodiments, the PNA sequence comprises between about 5 and 20bases. In some embodiments, the PNA sequence comprises between about 5and

-   -   bases. In some embodiments, the PNA sequence comprises between        about 5 and 10 bases. In some embodiments, the PNA sequence        comprises about 15 bases. In some embodiments, the PNA sequence        comprises about 10 bases.

In some embodiments, the ‘Specific binding entity’ is a primaryantibody. In some embodiments, the ‘Specific binding entity’ is asecondary antibody. In some embodiments, X is biotin. In someembodiments, m is 0, z is 0, and n is greater than 1. In someembodiments, n is an integer ranging from between 2 and 6. In someembodiments, ‘Linker’ comprises at least one PEG group.

In some embodiments, the ‘Specific Binding Entity’ is a primary antibodyand the PNA sequence comprises 10 nucleotides. In some embodiments, the‘Specific Binding Entity’ is a primary antibody and the PNA sequencecomprises at least two nucleotides having a substituent at the gammaposition.

In some embodiments, ‘Linker’ has the structure depicted in Formula(IVA):

wherein

d and e are integers each independently ranging from 2 to 20;

Q is a bond, O, S, or N(R^(c))(R^(d));

R^(a) and R^(b) are independently H, a C₁-C₄ alkyl group, F, Cl, orN(R^(c))(R^(d));

R^(c) and R^(d) are independently CH3 or H; and

A and B are independently a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated group havingbetween 1 and 12 carbon atoms and optionally having one or more O, N, orS heteroatoms.

In some embodiments, d and e are integers ranging from 2 to 6. In someembodiments, at least one of A or B comprises a cleavable moiety. Insome embodiments, the cleavable moiety is a photocleavable group. Insome embodiments, the cleavable moiety is a chemically cleavable group.In some embodiments, ‘Specific binding entity’ is an antibody, ‘Linker’comprises at least one PEG group, m is 0, z is 0, and n is greaterthan 1. In some embodiments, ‘Specific binding entity’ is an antibody,‘Linker’ comprises at least one PEG group, and n is greater than 1. Insome embodiments, ‘Linker’ further comprises at least one cleavablegroup. In some embodiments, X is a hapten.

In another aspect of the present disclosure is an oligomer having thestructure of Formula (III):

wherein

T is a group having between 1 and 4 carbon atoms and optionallysubstituted with O, N, or S and having a terminal reactive moiety;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

‘PNA’ is a PNA sequence including at least one nucleotide having asubstituent at a gamma carbon position;

X is a label;

Y is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms;

m is 0 or an integer ranging from 1 to 6; and

z is 0 or 1.

In some embodiments, X is biotin, an enzyme, a chromogen, a fluorophore,a hapten, and a mass spectrometry tag.

In some embodiments, X is biotin.

In some embodiments, the PNA sequence has at least two gamma PNA bases.In some embodiments, each of the at least two gamma PNA bases issubstituted with the same amino acid, miniPEG, peptide, or polymer. Insome embodiments, each of the at least two gamma PNA bases issubstituted with a different amino acid, miniPEG, peptide, or polymer.In some embodiments, the amino acid, miniPEG, peptide, or polymerincludes at least one reporter moiety (e.g. a fluorophore, a chromogen).In some embodiments, the PNA sequence has at least three gamma PNAbases. In some embodiments, the PNA sequence has at least four gamma PNAbases.

In some embodiments, the at least one substituted nucleotide of the PNAsequence includes a lysine residue, a peptide, or a miniPEG, where eachof the lysine residue, a peptide, or a miniPEG may have one or morereporter moieties coupled thereto.

In some embodiments, the PNA sequence, including PNA sequences havingone or more substituents at a gamma position, comprises between about 5and 20 bases. In some embodiments, the PNA sequence, including PNAsequences having one or more substituents at a gamma position, comprisesbetween about 5 and 15 bases. In some embodiments, the PNA sequence,including PNA sequences having one or more substituents at a gammaposition, comprises between about 5 and 10 bases. In some embodiments,the PNA sequence, including PNA sequences having one or moresubstituents at a gamma position, comprises about 15 bases. In someembodiments, the PNA sequence, including PNA sequences having one ormore substituents at a gamma position, comprises about 10 bases.

In some embodiments, the ‘Specific Binding Entity’ is a primary antibodyand the PNA sequence comprises 10 nucleotides. In some embodiments, the‘Specific Binding Entity’ is a primary antibody and the PNA sequenceincludes at least one substituent at one gamma position.

In some embodiments, m is 0, z is 0, and n is greater than 1.

In some embodiments, n is an integer ranging from between 2 and 6. Insome embodiments, ‘Linker’ comprises at least one PEG group. In someembodiments, ‘Linker’ has the structure depicted in Formula (IVA):

wherein

d and e are integers each independently ranging from 2 to 20;

Q is a bond, O, S, or N(R^(c))(R^(d));

R^(a) and R^(b) are independently H, a C₁-C₄ alkyl group, F, Cl, orN(R^(c))(R^(d));

R^(c) and R^(d) are independently CH₃ or H; and

A and B are independently a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated group havingbetween 1 and 12 carbon atoms and optionally having one or more O, N, orS heteroatoms.

In some embodiments, d and e are integers ranging from 2 to 6. In someembodiments, at least one of A or B comprises a cleavable moiety. Insome embodiments, the cleavable moiety is a photocleavable group. Insome embodiments, the cleavable moiety is a chemically cleavable group.

In another aspect of the present disclosure is a method of detecting atarget in a sample, comprising:

(a) contacting the sample with a first conjugate, the first conjugatehaving the structure of Formula (IA):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody, an antibody fragment, a drug/antibody complex, and a nucleicacid;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

Z is selected from the group consisting of a PNA sequence including atleast one nucleotide having a substituent at a gamma carbon position, anuncharged DNA sequence, and a DNA sequence comprising charged anduncharged bases;

X is a label;

Y is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms;

m is 0 or an integer ranging from 1 to 6;

z is 0 or 1; and

n is an integer ranging from 1 to 12; and

(b) contacting the sample with first detection reagents to facilitatedetection of the first conjugate.

In some embodiments, the method further comprises contacting the samplewith a second conjugate of Formula (IA), wherein the first and secondconjugates are different (e.g. include a different specific bindingentity and/or a different label).

In some embodiments, X is selected from the group consisting of biotin,an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometrytag.

In some embodiments, the ‘Specific Binding Entity’ is a primary antibodyand Z is a PNA sequence or gamma PNA sequence. In some embodiments, the‘Specific Binding Entity’ is a primary antibody and Z is a PNA sequencehaving 10 nucleotides. In some embodiments, the ‘Specific BindingEntity’ is a primary antibody and Z is a gamma PNA sequence.

In some embodiments, Z comprises a DNA sequence comprising onlyuncharged DNA bases. In some embodiments, Z comprises a DNA sequencecomprising a mixture of charged and uncharged bases. In someembodiments, Z comprises a DNA sequence in which at least 50% of thebases in the DNA sequence are uncharged.

In some embodiments, the PNA sequence has at least two gamma PNA bases.In some embodiments, each of the at least two gamma PNA bases issubstituted with the same amino acid, miniPEG, peptide, or polymer. Insome embodiments, each of the at least two gamma PNA bases issubstituted with a different amino acid, miniPEG, peptide, or polymer.In some embodiments, the amino acid, miniPEG, peptide, or polymerincludes at least one reporter moiety (e.g. a fluorophore, a chromogen).In some embodiments, the PNA sequence has at least three gamma PNAbases. In some embodiments, the PNA sequence has at least four gamma PNAbases.

In some embodiments, the PNA sequence comprises between about 5 and 20bases. In some embodiments, the PNA sequence comprises between about 5and 15 bases. In some embodiments, the PNA sequence comprises betweenabout 5 and 10 bases. In some embodiments, the PNA sequence comprisesabout 15 bases. In some embodiments, the PNA sequence comprises about 10bases.

In another aspect of the present disclosure is a method of detecting atarget in a sample, comprising:

(a) contacting the sample with a first PNA conjugate, the first PNAconjugate having the structure of Formula (IIB):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody, an antibody fragment, a drug/antibody complex, and a nucleicacid;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

‘PNA’ is a PNA sequence including at least one nucleotide having asubstituent at a gamma carbon position;

X is a label;

Y is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms;

m is 0 or an integer ranging from 1 to 6;

z is 0 or 1; and

n is an integer ranging from 1 to 12; and

(b) contacting the sample with first detection reagents to facilitatedetection of the PNA conjugate.

In some embodiments, the method further comprises contacting the samplewith a second PNA conjugate (e.g. another conjugate of Formula (JIB)),wherein the first PNA conjugate and the second PNA conjugate aredifferent, i.e. have at least one component or moiety which isdifferent.

In some embodiments, X is selected from the group consisting of biotin,an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometrytag.

In some embodiments, the PNA sequence has at least two gamma PNA bases.In some embodiments, each of the at least two gamma PNA bases issubstituted with the same amino acid, miniPEG, peptide, or polymer. Insome embodiments, each of the at least two gamma PNA bases issubstituted with a different amino acid, miniPEG, peptide, or polymer.In some embodiments, the amino acid, miniPEG, peptide, or polymerincludes at least one reporter moiety (e.g. a fluorophore, a chromogen).In some embodiments, the PNA sequence has at least three gamma PNAbases. In some embodiments, the PNA sequence has at least four gamma PNAbases.

In some embodiments, the PNA sequence comprises between about 5 and 20bases. In some embodiments, the PNA sequence comprises between about 5and 15 bases. In some embodiments, the PNA sequence comprises betweenabout 5 and 10 bases. In some embodiments, the PNA sequence comprisesabout 15 bases. In some embodiments, the PNA sequence about 10 bases.

In some embodiments, the ‘Specific binding entity’ is a primary antibodyand wherein the primary antibody is specific to a first target. In someembodiments, the ‘Specific binding entity’ is a secondary antibody, andwherein the method further comprises the step of contacting the samplewith a primary antibody specific for a first target prior to contactingthe sample with the first conjugate, and wherein the first conjugate isspecific to the first primary antibody. In some embodiments, the firstdetection reagents are anti-label antibodies specific to a label of theconjugate. In some embodiments, the label is a hapten and the anti-labelantibodies are anti-hapten antibodies. In some embodiments, thedetection reagents comprise a PNA, gamma PNA or DNA sequencecomplementary to a PNA sequence or gamma PNA sequence of the firstconjugate, the complementary PNA, gamma PNA or DNA sequence conjugatedto a reporter moiety. In some embodiments, the reporter moiety is afluorophore. In some embodiments, the reporter moiety is a chromogen. Insome embodiments, the reporter moiety is an enzyme. In some embodiments,the reporter moiety is a hapten, and where the method further comprisescontacting the sample with anti-hapten antibodies specific to the haptenof the complementary PNA, gamma PNA or DNA sequence.

In another aspect of the present disclosure is a method of detecting atarget in a sample, comprising: (a) contacting the sample with a firstPNA conjugate, the first PNA conjugate having the structure of Formula(IIB):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody (e.g. a primary antibody or a secondary antibody), an antibodyfragment, a drug/antibody complex, and a nucleic acid;

‘Linker,’ including a cleavable group or moiety, that is a branched orunbranched, linear or cyclic, substituted or unsubstituted, saturated orunsaturated, group having between 2 and 80 carbon atoms, and optionallyhaving one or more heteroatoms selected from O, N, or S;

‘PNA’ is a PNA sequence including at least one nucleotide having asubstituent at a gamma carbon position;

X is a label;

Y is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms;

m is 0;

z is 0; and

n is an integer ranging from 1 to 12; and

(b) contacting the sample with a reagent (or a light source, dependingon the cleavable group selected) to cleave the cleavable group on the‘Linker;’ and (c) quantifying an amount of a cleaved ‘PNA’ sequence orgamma PNA sequence.

The skilled artisan will appreciate that the above-identified steps maybe repeated any number of times with different PNA conjugates (e.g. anyconjugates of Formula (IIB) to provide a multiplex assay.

In some embodiments, the ‘Specific Binding Entity’ is an antibody.

In some embodiments, the cleavable group is selected from the groupconsisting of a photocleavable group, a chemically cleavable group, oran enzymatically cleavable group. In some embodiments, the cleavablegroup is a disulfide bond.

In some embodiments, X is selected from the group consisting of biotin,an enzyme, a chromogen, a fluorophore, a hapten, and a mass spectrometrytag. In some embodiments, X is biotin.

In some embodiments, the quantification of the amount of the PNAsequence is performed using NanoString nCounter technology, such asdescribed herein. In some embodiments, the quantification of the amountof the PNA sequence is performed using Gyrolab technology, such asdescribed herein.

In some embodiments, after PNA cleavage, the antibody-bound tissuesection is re-stained with a conventional method such asimmunohistochemistry or immunofluorescence to allow visualization of thespatial distribution of the marker encoded by the PNA tag.

In some embodiments, the method further comprises introducing a singlestranded DNA or PNA sequence which is complementary to the PNA sequenceof the conjugate of Formula (IIB). In some embodiments, thecomplementary single stranded DNA or PNA sequence is conjugated to areporter moiety. In some embodiments, the complementary single strandedDNA or PNA sequence is conjugated to a hapten. In some embodiments, thecomplementary single stranded DNA or PNA sequence is conjugated todigoxigenin.

In some embodiments, the ‘Specific Binding Entity’ is a primaryantibody, and wherein the method further comprises introducing asecondary antibody specific for the primary antibody. In someembodiments, the secondary antibody is conjugated to a reporter moiety.

In some embodiments, the PNA sequence has at least two gamma PNA bases.In some embodiments, each of the at least two gamma PNA bases issubstituted with the same amino acid, miniPEG, peptide, or polymer. Insome embodiments, each of the at least two gamma PNA bases issubstituted with a different amino acid, miniPEG, peptide, or polymer.In some embodiments, the amino acid, miniPEG, peptide, or polymerincludes at least one reporter moiety (e.g. a fluorophore, a chromogen).In some embodiments, the PNA sequence has at least three gamma PNAbases. In some embodiments, the PNA sequence has at least four gamma PNAbases.

In some embodiments, the PNA sequence comprises between about 5 and 20bases. In some embodiments, the PNA sequence comprises between about 5and 15 bases. In some embodiments, the PNA sequence comprises betweenabout 5 and 10 bases. In some embodiments, the PNA sequence comprisesabout 15 bases. In some embodiments, the PNA sequence comprises about 10bases.

It is believed that since PNA contains no charges, unlike DNA, thebinding between PNA and DNA is stronger than that between DNA and DNA,allowing the use of comparatively shorter PNA sequences to labelantibodies while achieving binding affinity and specificity not possiblewith DNA-conjugates. It is also anticipated that the shorter PNAsequence may help minimize its interference with antibody-antigenbinding and tissue non-specific binding.

Therefore, the PNA-antibody conjugates disclosed herein are expected tomaintain the binding specificity of the antibody while the PNA oligomerfunctions as a unique molecular tag that can be either visualized insitu on slides (IF or IHC) through hybridization with a signalgenerating molecule, or quantified off the slides by a high-throughputtechnology such as NanoString technology, Gryos technology, or massspectrometry (see FIG. 1A). In addition, virtually unlimited uniquesequence/tags can be readily generated and detected under almostidentical conditions, eliminating the need to optimize individualconjugates. Importantly, a cleavable linker (either photo or chemicalcleavable) can be placed between the PNA oligomer and the antibody (seeFIG. 1A), allowing facile sample collection for off-slide proteinprofiling (multiplexed quantification).

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided to the Office upon request and thepayment of the necessary fee.

FIG. 1A provides an overview of the structure of a PNA conjugate andtheir use in both visualization of targets within a sample (qualitative)and quantitative assessment of the PNA tags cleaved from the boundantibody.

FIG. 1B provides the chemical structure of the heterobifunctionalcross-linker and PNA sequence (SEQ ID NO: 1).

FIG. 2A illustrates the characterization of an antibody andcorresponding PNA conjugate based on UV-Vis absorbance before PNAconjugation. Black and red trace represents two different antibodies asexamples.

FIG. 2B illustrates the characterization of an antibody andcorresponding PNA conjugate based on UV-Vis absorbance after PNAconjugation. Black and red trace represents two different antibodies asexamples.

FIG. 3A illustrates IHC detection of a biotinylated PNA. The figureillustrates tonsil slides treated with rabbit anti-CD45 antibodyfollowed by PNA-conjugated GAR, GAM, and GAM respectively. Biotin wasthen detected with SA-HRP and DAB deposition.

FIG. 3B illustrates IHC detection of a biotinylated PNA. The figureillustrates tonsil slides treated with mouse anti-Ki67 antibody followedby PNA-conjugated GAR, GAM, and GAM respectively. Biotin was thendetected with SA-HRP and DAB deposition.

FIG. 3C illustrates IHC detection of a biotinylated PNA. The figureillustrates tonsil slides treated with no primary antibody followed byPNA-conjugated GAR, GAM, and GAM, respectively. Biotin was then detectedwith SA-HRP and DAB deposition.

FIG. 3D illustrates a detection strategy using biotinylated PNA inaccordance with some embodiments of the disclosure.

FIG. 4A illustrates IHC detection of a biotinylated PNA as a membranemarker. Tonsil slides were treated with “haptenated” primary antibodies(i.e. an antibody labeled with a hapten) followed by treatment with acorresponding PNA-conjugate against the respective hapten. Biotin in thePNA conjugates was then detected with SA-HRP and DAB deposition.

FIG. 4B illustrates IHC detection of a biotinylated PNA as a nuclearmarker. Tonsil slides were treated with “haptenated” primary antibodiesfollowed by treatment with a corresponding PNA-conjugate against therespective hapten. Biotin in the PNA conjugates was then detected withSA-HRP and DAB deposition.

FIG. 5A illustrates the chemical cleavage of the PNA tag. A tonsil slidewas treated with a mouse anti-Ki67 antibody, and further treated withSA-HRP and DAB deposition.

FIG. 5B illustrates the chemical cleavage of the PNA tag. A tonsil slidewas treated with a mouse anti-Ki67 antibody, and further treated withabout 20 mM TCEP before SA-HRP incubation and DAB deposition.

FIG. 5C illustrates the chemical cleavage of the PNA tag. A tonsil slidewas treated with a rabbit anti-CD45 antibody, and further treated withSA-HRP and DAB deposition.

FIG. 5D illustrates the chemical cleavage of the PNA tag. A tonsil slidewas treated with a rabbit anti-CD45 antibody, and further treated withabout 20 mM TCEP before SA-HRP incubation and DAB deposition.

FIG. 6A illustrates fluorescence detection of the biotinylated PNA. Atonsil slide was treated with a mouse anti-Ki67 antibody followed byPNA-conjugated GAM. The slides were further incubated with SA-FITC.

FIG. 6B illustrates fluorescence detection of the biotinylated PNA. Atonsil slide was treated with no primary antibody followed byPNA-conjugated GAM. The slides were further incubated with SA-FITC.

FIG. 6C illustrates a fluorescent-based detection scheme utilizingbiotinylated PNA.

FIG. 7A sets forth a quantified measurement of antibody per slide basedon cleaved PNA-SA-FITC fluorescence intensity. Specifically, bar graphsshow the fluorescence intensity of PNA-SA-FITC cleaved from a tonsilslide treated with mouse anti-CD45, PNA-conjugated GAM, followed bySA-FITC and finally incubated with 20 mM TCEP. The control is a tonsilslide treated the same as the experiment but without primary antibody.

FIG. 7B sets forth a quantified measurement of antibody per slide basedon cleaved PNA-SA-FITC fluorescence intensity. Specifically, thestandard curve used to determine the concentration of PNA-SA-FITCcleaved from the experiment and control slides. Fluorescence intensitiesof sample and control are shown as mean±STDEV (N=3).

FIG. 8A illustrates PNA detection with fluorescently labeled complementDNA. Tonsil slides were treated with rabbit anti-Ki67 followed byPNA-conjugated GAR. The slides were then incubated with fluorescentlylabeled DNA sequences that are complement to the PNA tag. A FITC labeledDNA sequence was utilized.

FIG. 8B illustrates PNA detection with fluorescently labeled complementDNA. Tonsil slides not treated with a primary antibody and thus servedas a negative control. The slides were then incubated with fluorescentlylabeled DNA sequences that are complement to the PNA tag. A FITC labeledDNA sequence was utilized.

FIG. 8C illustrates PNA detection with fluorescently labeled complementDNA. A tonsil slide was treated with rabbit anti-Ki67 followed byPNA-conjugated GAR. The slides were then incubated with fluorescentlylabeled DNA sequences that are complement to the PNA tag. A rhodaminelabeled DNA sequence was utilized. Negative controls with no primaryantibody added with otherwise identical conditions were shown in FIG. 8Band FIG. 8D respectively.

FIG. 8D illustrates PNA detection with fluorescently labeled complementDNA. Tonsil slides not treated with a primary antibody and thus servedas a negative control. The slides were then incubated with fluorescentlylabeled DNA sequences that are complement to the PNA tag. A rhodaminelabeled DNA sequence was utilized.

FIG. 8E provides a schematic of the detection methods utilized ingenerating the images of FIGS. 8A, 8B, 8C, and 8D.

FIG. 9A illustrates chromogenic detection with complimentary haptenatedDNA. A tonsil slide was treated with primary mouse-anti-CD20 and thenincubated with PNA-conjugated GAM followed by hybridization withDIG-labeled DNA sequence that is compliment to the PNA tag. Finally, theslides were incubated with anti-DIG:HRP antibody and DAB deposition.

FIG. 9B illustrates chromogenic detection with complimentary haptenatedDNA. A tonsil slide was treated with rabbit-anti-Ki67 and then incubatedwith PNA-conjugated GAM followed by hybridization with DIG-labeled DNAsequence that is compliment to the PNA tag. Finally, the slides wereincubated with anti-DIG:HRP antibody and DAB deposition.

FIG. 9C illustrates tonsil tissue that was treated in the same manner asthe tissue represented in FIGS. 9A and 9B, but with no primaryantibodies, thus serving as a negative control.

FIG. 9D sets forth a schematic of a detection scheme utilized ingenerating the images of FIGS. 9A, 9B, and 9C.

FIG. 10 illustrates a possible synthetic scheme of the photocleavableheterobifunctional cross-linker.

FIG. 11 illustrates the chemical structure of the photo-labile linkerused to synthesize photocleavable PNA.

FIG. 12A illustrates the photocleavage of PNA. Slides were treated withKi67, then GAR-PL-PNA. The slide was irradiated with a hand-held UV lampfor 0 minutes.

FIG. 12B illustrates the photocleavage of PNA. Slides were treated withKi67, then GAR-PL-PNA. The slide was irradiated with a hand-held UV lampfor 5 minutes.

FIG. 12C illustrates the photocleavage of PNA. Slides were treated withKi67, then GAR-PL-PNA. The slide was irradiated with a hand-held UV lampfor 10 minutes.

FIG. 13 illustrates the selective PNA photocleavage. Tonsil slide wastreated with Ki67, then GAR-PL-PNA. The -indicated area was thenirradiated with UV light from a laser capture microdissectioninstrument. LCM After washing the slide to remove the cleaved PNA, thestaining was completed with SA-HRP and DAB detection. A UV irradiatedgerminal center lost color compared to non-irradiated germinal center onthe same slide.

FIG. 14A sets forth a flowchart outlining various methods of detectingtargets in a sample using the PNA conjugates of the present disclosure.

FIG. 14B sets forth a flowchart outlining various methods of detectingtargets in a sample using the PNA conjugates of the present disclosure.

FIG. 14C sets forth a flowchart outlining various methods of detectingtargets in a sample using the PNA conjugates of the present disclosure.

FIG. 15 illustrates the stability of the PNA antibody conjugates. Tonsilslides were incubated with primary antibodies (anti-CD45 and anti-Ki67)followed by GAM-PNA and GAR-PNA respectively and detected with SA-HRPand DAB deposition. The same antibody-PNA conjugates were used at latertime as indicated to assess the conjugates stability.

FIG. 16 illustrates the difference between PNA having a substituent at agamma-carbon (indicated by the “R” group, which is non-limiting) and aPNA which does not have a substituent at the gamma-carbon.

FIG. 17 illustrates a branched PNA sequence. The main PNA sequence(depicted in red) has 4 substituted PNA bases. The substituents of themain PNA sequence are gamma-PNA sequences (depicted in black). Each PNAsequence has 4 substituents. These substituents could be, shown asincluding four gamma PNA nucleotides, each substituted with a miniPEG orother reporter moieties. Each miniPEG may comprise one or more labels orreporter moieties.

FIG. 18 is a schematic illustrating the conjugation of an antibody to aPNA oligomer, the coupling utilizing “click chemistry.” Here, a reducedantibody is functionalized with a DBCO group and then coupled to a PNAoligomer comprising an azide group. The PNA oligomer may comprise one ormore nucleotides having a substituent at a gamma carbon.

FIG. 19 illustrates tonsil tissue incubated with anti-Ki67 primaryantibody (Rabbit mAb) and GAR-PNA (conjugated with click chemistry)secondary antibody and detected with SA-HRP. The image shows that thePNA was successfully conjugated via click chemistry to the Antibody.

FIG. 20 is a schematic illustrating the conjugation of an antibody to aPNA oligomer via maleimide moiety.

FIG. 21A illustrates tonsil tissue incubated with anti-Ki67 then aGAR-short PNA (sequence shown in previous slide) and detected withSA-HRP. Detection of the biotin on the short PNA sequence. The imagesshow that short PNA is successfully conjugated on the Ab.

FIG. 21B illustrates tonsil tissue incubated with anti-Ki67 thenGAR-HRP. The images show that short PNA is successfully conjugated onthe Ab. This is used to compare standard detection (FIG. 21B) toGAR-short PNA based detection (FIG. 21A).

FIG. 22A illustrates tonsil incubated with CD3-shortPNA and detectedwith SA-HRP. IHC of shortPNA conjugated with primary antibody. Theconcentration of the Ab-shortPNA conjugate used was 5 μg/mL (PNAsequence: Biotin-o-CCATCTTCAG-Lys(SMCC)sequence) (SEQ ID NO: 19).

FIGS. 22B, 22C, and 22D illustrate tonsil incubated with second, third,and fourth examples of primary antibody-short PNA conjugates(CD8-shortPNA, CD34-shortPNA, and Ki67-shortPNA respectively).

FIG. 23 illustrates chromogenic staining with CD3 conjugated to (10bases) PNA sequences referred to as sPNA2 having the sequence:Biotin-o-TTAGTCCAAC-Lys(SMCC) (SEQ ID NO: 20). Tonsil tissue wasstaining with the PNA-conjugated Ab followed by SA-HRP to target thebiotin moiety on the PNA sequence. The staining is localized provingthat the PNA conjugation did not alter the Ab functionality

FIG. 24 illustrates chromogenic staining with CD8 conjugated to sPNA2(10 bases) where the PNA sequence was Biotin-o-CCATCTTCAG-Lys(C₆SH) (SEQID NO: 21). Tonsil tissue was staining with the PNA-conjugated Abfollowed by SA-HRP to target the biotin moiety on the PNA sequence. Thestaining is localized proving that the PNA conjugation did not alter theAb functionality

FIG. 25 illustrates tonsil tissue incubated with anti-Ki67 and GAR-shortPNA followed by DNA-DIG (DNA sequence complementary to the short PNA tagcalled AB14 that has a DIG on its end). antiDIG-HRP antibody was addedfor detection. In this case, DNA was detected that was complementary tothe PNA tag. Two different concentrations of the complementary DNA-DIGare shown.

FIG. 26 illustrates fluorescent duplexing via DNA conjugation. Thefigure further illustrates detection through hybridization but this timethrough florescence. The complementary DNA includes one or morefluorescent tags. Two primary antibodies CD3 and CD8, are conjugated,respectively, to two different short PNA tags. Each of the twocomplementary DNA sequences have a fluorophore (SEQ ID NOS: 4, 5, 11).

FIGS. 27A, 27B, and 27C illustrate tonsil tissue slide incubated with amixture of anti-CD3-shortPNA1 and anti-CD8-shortPNA2 (anti-CD3 andantiCD-8 conjugated to short PNA1 and short PNA2 respectively) followedby incubation with 2 DNA sequences complementary to sPNA1 and sPNA2respectively referred to as AB15 and AB19. AB15 has AlexaFluor 488 onits end, while AB19 has a AlexaFluor 647 on its end. The twofluorescently-labeled DNA sequences binds to their complement PNAsequences on tissue to generate a fluorescently stained tissue slide.The fluorescently stained tissue is imaged with a fluorescentmicroscope. The green channel (FIG. 27A) shows AlexaFluor 488 on the CD3positive cells. The Red channel (FIG. 27B) shows the AlexaFluor 647 onthe CD8 positive cells at the same field of view. A merge of the twoimages (FIG. 27C) shows that all color red cells (CD8 positive) are alsogreen (CD3 positive) because all the cells that express CD8 do expressCD3. Some green cells are not red because not all CD3 cells are CD8. CD8is s subpopulation of CD3.

FIG. 28 illustrates tonsil tissue incubated with anti-Ki67 (Rabbit mAb)followed by GAR-gamma-PNA (goat anti rabbit conjugated to gamma-PNA) andfinally streptavidin-HRP which will bind to the biotin at the end of thegamma-PNA. The signal was as expected, and no non-specific signal wasobserved.

FIG. 29A illustrates tonsil tissue incubated with a primary antibodyconjugated to gamma-PNA and detected with SA-HRP. Differentconcentrations of the CD3-gamma-PNA (10 μg/mL, 5 μg/mL, 1 μg/mL) areshown with 2 magnifications (20× and 40×). This shows that a primaryantibody can be also conjugated to gamma PNA and detected directly.

FIG. 29B provides another example of a primary antibody (CD8) conjugatedto a gamma PNA at different concentrations (10 μg/mL, 5 μg/mL, 1 μg/mL).

FIG. 29C provides another example of a primary antibody (PD-L1)conjugated to a gamma-PNA at different concentrations (1.5 μg/mL, 1μg/mL, 0.5 μg/mL).

FIG. 30 illustrates the UV absorbance of GAR-PNA and GAR-gamma PNAconjugates. Here, the antibody absorbs light primarily at 280 nm;whereas PNA absorbs light primarily mainly at 260 nm. When PNA isconjugated to an antibody, the absorbance at 260 increases. The ratio260 nm/280 nm is used to assess the conjugation efficiency. An increasein the 260 nm/280 nm ratio indicates a higher PNA loading, which isexpected as the gamma-PNA is more hydrophilic than PNA without any gammasubstituent. The 260 nm/280 nm ratio of non-conjugated antibody (pureantibody) is around 0.58 which increases to 1.1 when conjugated to PNAand to 1.3 when conjugated to gamma-PNA.

FIG. 31 illustrates the UV absorbance of GAR, CD3 and CD8-shortPNAconjugates with 260/280 ratios. It is shown that the 260 nm/280 nm ratioincreases as PNA is conjugated to the antibody.

FIG. 32 illustrates the principle of using use of Gryos technology forthe quantification of PNA.

FIG. 33 provides a schematic illustrating the steps of quantification ofcleaved PNA tag using Gryos technology, and re-staining the slide by IHCto allow visualization of the same marker encoded by the PNA tag.

FIG. 34A illustrates IHC staining of the protein target followingcleavage of a PNA sequence or gamma PNA sequence from a PNA-antibodyconjugate.

FIG. 34B illustrates IHC staining of the protein target followingcleavage of a PNA sequence or gamma PNA sequence from a PNA-antibodyconjugate.

FIG. 35A provides a comparison of the detection of different PNAoligomers and DNA oligomers using Gyros technology.

FIG. 35B illustrates the signal-to-background (S/B) ratio of the PNAdetected using Gyros technology.

FIG. 36A illustrates tonsil stained with Ki67 (rabbit Ab) then GoatAnti-Rabbit (GAR) conjugated to a PNA sequence (CCATCTTCAG) (SEQ ID NO:4). The absence of the signal (as shown here) indicated that at theexperimental conditions (200 nM of DNA, 37° C., 30 min) the DNA did nothybridize. The DNA PNA affinity is not strong enough to assurehybridization with such short PNA sequence at the experimentalconditions.

FIG. 36B illustrates tonsil stained with Ki67 (rabbit Ab) then GoatAnti-Rabbit (GAR) conjugated to a gamma PNA sequence. The DNA gamma PNAaffinity is believed to be strong enough to assure hybridization, thusgenerating the brown signal (SEQ ID NO: 8).

DETAILED DESCRIPTION

In general, the present disclosure is directed to conjugates, e.g. PNAconjugates, as well as methods of employing the conjugates for detectingone or more targets in a biological sample, e.g. a tissue sample.Without wishing to be bound by any particular theory, it is believedthat the conjugates, when used in an assay, allow for qualitative andquantitative evaluation of a high numbers of targets, including proteintargets, simultaneously (see FIG. 1A).

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless the context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of or “exactly one of,” or, when used inthe claims, “consisting of,” will refer to the inclusion of exactly oneelement of a number or list of elements. In general, the term “or” asused herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

The terms “comprising,” “including,” “having,” and the like are usedinterchangeably and have the same meaning. Similarly, “comprises,”“includes,” “has,” and the like are used interchangeably and have thesame meaning. Specifically, each of the terms is defined consistent withthe common United States patent law definition of “comprising” and istherefore interpreted to be an open term meaning “at least thefollowing,” and is also interpreted not to exclude additional features,limitations, aspects, etc. Thus, for example, “a device havingcomponents a, b, and c” means that the device includes at leastcomponents a, b and c. Similarly, the phrase: “a method involving stepsa, b, and c” means that the method includes at least steps a, b, and c.Moreover, while the steps and processes may be outlined herein in aparticular order, the skilled artisan will recognize that the orderingsteps and processes may vary.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

As used herein, the term “alkyl” refers to a straight or branchedhydrocarbon chain that comprises a fully saturated (no double or triplebonds) hydrocarbon group. The alkyl group may be substituted orunsubstituted.

As used herein, the term “antibody,” refers to immunoglobulins orimmunoglobulin-like molecules, including by way of example and withoutlimitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, andsimilar molecules produced during an immune response in any vertebrate,(e.g., in mammals such as humans, goats, rabbits and mice) and antibodyfragments (such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragmentsand Fab fragments as are known in the art, recombinant antibodyfragments (such as sFv fragments, dsFv fragments, bispecific sFvfragments, bispecific dsFv fragments, F(ab)′2 fragments, single chain Fvproteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies,and triabodies (as are known in the art), and camelid antibodies) thatspecifically bind to a molecule of interest (or a group of highlysimilar molecules of interest) to the substantial exclusion of bindingto other molecules. Antibody further refers to a polypeptide ligandcomprising at least a light chain or heavy chain immunoglobulin variableregion which specifically recognizes and binds an epitope of an antigen.Antibodies may be composed of a heavy and a light chain, each of whichhas a variable region, termed the variable heavy (VH) region and thevariable light (VL) region. Together, the VH region and the VL regionare responsible for binding the antigen recognized by the antibody. Theterm antibody also includes intact immunoglobulins and the variants andportions of them well known in the art.

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may be specifically bound by the products of specifichumoral or cellular immunity, such as an antibody molecule or T-cellreceptor. Antigens can be any type of molecule including, for example,haptens, simple intermediary metabolites, sugars (e.g.,oligosaccharides), lipids, and hormones as well as macromolecules suchas complex carbohydrates (e.g., polysaccharides), phospholipids, nucleicacids and proteins.

As used herein, the terms “biological sample” or “tissue sample” are anysolid or fluid sample obtained from, excreted by or secreted by anyliving organism, including without limitation, single celled organisms,such as bacteria, yeast, protozoans, and amoebas among others,multicellular organisms (such as plants or animals, including samplesfrom a healthy or apparently healthy human subject or a human patientaffected by a condition or disease to be diagnosed or investigated, suchas cancer). For example, a biological sample can be a biological fluidobtained from, for example, blood, plasma, serum, urine, bile, ascites,saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodilysecretion, a transudate, an exudate (for example, fluid obtained from anabscess or any other site of infection or inflammation), or fluidobtained from a joint (for example, a normal joint or a joint affectedby disease). A biological sample can also be a sample obtained from anyorgan or tissue (including a biopsy or autopsy specimen, such as a tumorbiopsy) or can include a cell (whether a primary cell or cultured cell)or medium conditioned by any cell, tissue or organ. The samples may betumor samples, including those from melanoma, renal cell carcinoma, andnon-small-cell lung cancers. In some embodiments, the samples areanalyzed for disease states such as cancer by detecting targets,including biomarkers (e.g. proteins or nucleic acid sequences), withinthe tissue sample. The described embodiments of the disclosed method canalso be applied to samples that do not have abnormalities, diseases,disorders, etc., referred to as “normal” samples or “control” samples.For example, it may be useful to test a subject for cancer by takingtissue samples from multiple locations, and these samples may be used ascontrols and compared to later samples to determine whether a particularcancer has spread beyond its primary origin. In some embodiments, thesample may be a protein that has been extracted and purified from a celllysate, a tissue, or from a whole organism through the conventionalprotein extraction methods. In the context of the present disclosure,the extracted proteins can then be mixed with PNA-conjugated antibodieswhere the antibodies become bound to the specific proteins. The PNA isthen released and counted to quantify the protein.

As used herein, “C_(a) to C_(b)” in which “a” and “b” are integers referto the number of carbon atoms in an alkyl, alkenyl or alkynyl group, orthe number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl,cycloalkynyl or aryl group, or the total number of carbon atoms andheteroatoms in a heteroalkyl, heterocyclyl, heteroaryl orheteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, ring of thecycloalkyl, ring of the cycloalkenyl, ring of the cycloalkynyl, ring ofthe aryl, ring of the heteroaryl or ring of the heteroalicyclyl cancontain from “a” to “b”, inclusive, carbon atoms. Thus, for example, a“C₁ to C₄ alkyl” group refers to all alkyl groups having from 1 to 4carbons, that is, CH₃—, CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—,CH₃CH₂CH(CH₃)— and (CH₃)₃C—. If no “a” and “b” are designated withregard to an alkyl, alkenyl, alkynyl, cycloalkyl cycloalkenyl,cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group, the broadestrange described in these definitions is to be assumed.

As used herein, “conjugate” refers to two or more molecules (and/ormaterials such as nanoparticles) that are covalently linked into alarger construct.

As used herein, the term “couple” or “coupling” refers to the joining,bonding (e.g. covalent bonding), or linking of one molecule or atom toanother molecule or atom.

As used herein, the term “detection probes” include nucleic acid probesor primary antibodies which bind to specific targets (e.g. nucleic acidsequences, proteins, etc.). The detection probes may include a label fordirect detection, such as radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens(including, but not limited to, DNP), and enzymes. Alternatively, thedetection probes may contain no label or tag and may be detectedindirectly (e.g. with a secondary antibody that is specific for thedetection probe).

As used herein, the terms “gamma PNA” and “gamma PNA sequence” refer toany PNA sequencing having at least one nucleotide that comprises asubstituent at a gamma position, i.e. at a gamma carbon. FIG. 16illustrates the difference between a PNA nucleotide and one having asubstituent at the gamma-carbon position, where the “R” group is forillustrative purposes only and is non-limiting.

As used herein, the term “haptens” are small molecules that can combinespecifically with an antibody, but typically are substantially incapableof being immunogenic except in combination with a carrier molecule. Insome embodiments, haptens include, but are not limited to, pyrazoles(e.g. nitropyrazoles); nitrophenyl compounds; benzofurazans;triterpenes; ureas (e.g. phenyl ureas); thioureas (e.g. phenylthioureas); rotenone and rotenone derivatives; oxazole (e.g. oxazolesulfonamides); thiazoles (e.g. thiazole sulfonamides); coumarin andcoumarin derivatives; and cyclolignans. Additional non-limiting examplesof haptens include thiazoles; nitroaryls; benzofurans; triperpenes; andcyclolignans. Specific examples of haptens include di-nitrophenyl,biotin, digoxigenin, and fluorescein, and any derivatives or analogsthereof. Other haptens are described in U.S. Pat. Nos. 8,846,320;8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of whichare incorporated herein by reference in their entirety. The haptensthemselves may be suitable for direct detection, i.e. they may give offa suitable signal for detection.

As used herein, the terms “halogen atom” or “halogen” mean any one ofthe radio-stable atoms of column 7 of the Periodic Table of theElements, such as, fluorine, chlorine, bromine and iodine.

As used herein, the term “immunohistochemistry” refers to a method ofdetermining the presence or distribution of an antigen in a sample bydetecting interaction of the antigen with a specific binding agent, suchas an antibody. A sample is contacted with an antibody under conditionspermitting antibody-antigen binding. Antibody-antigen binding can bedetected by means of a detectable label conjugated to the antibody(direct detection) or by means of a detectable label conjugated to asecondary antibody, which binds specifically to the primary antibody(indirect detection).

As used herein, the terms “multiplex,” “multiplexed,” or “multiplexing”refer to detecting multiple targets in a sample concurrently,substantially simultaneously, or sequentially. Multiplexing can includeidentifying and/or quantifying multiple distinct nucleic acids (e.g.,DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) bothindividually and in any and all combinations.

As used herein, the term “oligonucleotides,” “polynucleotides” and“nucleic acids” are used here to encompass all forms of nucleic acidmolecules. Without limitation, this category includes ribonucleic acids(RNA), deoxyribonucleic acid (DNA), peptide nucleic acids (PNA), andtheir derivatives, with and without modifications, respectively.

As used herein, the term “primary antibody” refers to an antibody whichbinds specifically to a target protein antigen in a tissue sample. Aprimary antibody is generally the first antibody used in animmunohistochemical procedure. Primary antibodies may thus serve as“detection probes” for detecting a target within a tissue sample.

As used herein, the term “peptide nucleic acids” or “PNAs” areoligonucleotide analogues in which the sugar-phosphate backbone has beenreplaced by a pseudopeptide skeleton. PNAs bind DNA and RNA with highspecificity and selectivity, leading to PNA-RNA and PNA-DNA hybridswhich are believed to be more stable than the corresponding nucleic acidcomplexes. The binding affinity and selectivity of PNAs for nucleicacids can be modified by the introduction of stereogenic centers (suchas D-Lys-based units) into the PNA backbone. PNA can be an oligomer,linked polymer or chimeric oligomer. Methods for the chemical synthesisand assembly of PNAs are described in U.S. Pat. Nos. 5,539,082,5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, and 5,786,571,the disclosure of which are hereby incorporated by reference herein intheir entireties. The term “PNA” as used throughout, includes PNAsequences having one or more bases which are substituted at a gammaposition (“gamma PNA”), i.e. the term PNA or PNA sequence includes gammaPNA or gamma PNA sequences.

As used herein, the terms “reactive group” “reactive groups” as usedthroughout mean any of a variety of groups (e.g. functional groups)suitable for coupling a first unit to a second unit as described herein.For example, the reactive group might be an amine-reactive group, suchas an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, anacid chloride, such as sulfonyl chloride, aldehydes and glycols,epoxides and oxiranes, carbonates, arylating agents, imidoesters,carbodiimides, anhydrides, and combinations thereof. Suitablethiol-reactive functional groups include haloacetyl and alkyl halides,maleimides, aziridines, acryloyl derivatives, arylating agents,thiol-disulfide exchange reagents, such as pyridyl disulfides,TNB-thiol, and disulfide reductants, and combinations thereof. Suitablecarboxylate-reactive functional groups include diazoalkanes, diazoacetylcompounds, carbonyldiimidazole compounds, and carbondiimides. Suitablehydroxyl-reactive functional groups include epoxides and oxiranes,carbonyldiimidazole, N,N′-disuccinimidyl carbonates orN-hydroxysuccinimidyl chloroformates, periodate oxidizing compounds,enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde andketone-reactive functional groups include hydrazines, Schiff bases,reductive amination products, Mannich condensation products, andcombinations thereof. Active hydrogen-reactive compounds includediazonium derivatives, Mannich condensation products, iodinationreaction products, and combinations thereof. Photoreactive chemicalfunctional groups include aryl azides, halogenated aryl azides,benzophonones, diazo compounds, diazirine derivatives, and combinationsthereof.

As used herein, the phrase “reporter moiety” refers to a molecule ormaterial that can produce a detectable (such as visually, electronicallyor otherwise) signal that indicates the presence (i.e. qualitativeanalysis) and/or concentration (i.e. quantitative analysis) of theconjugate, including PNA conjugate, in a sample. A detectable signal canbe generated by any known or yet to be discovered mechanism includingabsorption, emission and/or scattering of a photon (including radiofrequency, microwave frequency, infrared frequency, visible frequencyand ultra-violet frequency photons).

As used herein, the term “secondary antibody” herein refers to anantibody which binds specifically to a detection probe or portionthereof (e.g. a hapten or a primary antibody), thereby forming a bridgebetween the detection probe and a subsequent reagent (e.g. a label, anenzyme, etc.), if any. A secondary antibody may be used to indirectlydetect the detection probes, e.g. primary antibodies. Examples ofsecondary antibodies include anti-tag antibodies, anti-speciesantibodies, and anti-label antibodies, each described herein.

As used herein the term “specific binding entity” refers to a member ofa specific-binding pair. Specific binding pairs are pairs of moleculesthat are characterized in that they bind each other to the substantialexclusion of binding to other molecules (for example, specific bindingpairs can have a binding constant that is at least 10³ M⁻¹ greater, 10⁴M⁻¹ greater or 10¹ M⁻¹ greater than a binding constant for either of thetwo members of the binding pair with other molecules in a biologicalsample). Examples of specific binding moieties include specific bindingproteins (for example, antibodies, lectins, avidins such asstreptavidins, and protein A). Specific binding moieties can alsoinclude the molecules (or portions thereof) that are specifically boundby such specific binding proteins. Specific binding entities includeprimary antibodies, described above, or nucleic acid probes.

As used herein, the terms “stain,” “staining,” or the like as usedherein generally refers to any treatment of a biological specimen thatdetects and/or differentiates the presence, location, and/or amount(such as concentration) of a particular molecule (such as a lipid,protein or nucleic acid) or particular structure (such as a normal ormalignant cell, cytosol, nucleus, Golgi apparatus, or cytoskeleton) inthe biological specimen. For example, staining can provide contrastbetween a particular molecule or a particular cellular structure andsurrounding portions of a biological specimen, and the intensity of thestaining can provide a measure of the amount of a particular molecule inthe specimen. Staining can be used to aid in the viewing of molecules,cellular structures and organisms not only with bright-fieldmicroscopes, but also with other viewing tools, such as phase contrastmicroscopes, electron microscopes, and fluorescence microscopes. Somestaining performed by the system 2 can be used to visualize an outlineof a cell. Other staining performed by the system 2 may rely on certaincell components (such as molecules or structures) being stained withoutor with relatively little staining other cell components. Examples oftypes of staining methods performed by the system 2 include, withoutlimitation, histochemical methods, immunohistochemical methods, andother methods based on reactions between molecules (includingnon-covalent binding interactions), such as hybridization reactionsbetween nucleic acid molecules. Particular staining methods include, butare not limited to, primary staining methods (e.g., H&E staining, Papstaining, etc.), enzyme-linked immunohistochemical methods, and in situRNA and DNA hybridization methods, such as fluorescence in situhybridization (FISH).

Whenever a group or moiety is described as being “substituted” or“optionally substituted” (or “optionally having” or “optionallycomprising”) that group may be unsubstituted or substituted with one ormore of the indicated substituents. Likewise, when a group is describedas being “substituted or unsubstituted” if substituted, thesubstituent(s) may be selected from one or more the indicatedsubstituents. If no substituents are indicated, it is meant that theindicated “optionally substituted” or “substituted” group may besubstituted with one or more group(s) individually and independentlyselected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,(heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy,acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protectedC-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro,silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy,trihalomethanesulfonyl, trihalomethanesulfonamido, an amino, ether,amino (e.g. a mono-substituted amino group or a di-substituted aminogroup), and protected derivatives thereof. Any of the above groups mayinclude one or more heteroatoms, including O, N, or S. For example,where a moiety is substituted with an alkyl group, that alkyl group maycomprise a heteroatom selected from O, N, or S (e.g.—(CH₂—CH₂—O—CH₂—CH₂)—).

As used herein, the term “substantially” means the qualitative conditionof exhibiting total or near-total extent or degree of a characteristicor property of interest. In some embodiments, “substantially” meanswithin about 20%. In some embodiments, “substantially” means withinabout 15%. In some embodiments, “substantially” means within about 10%.In some embodiments, “substantially” means within about 5%.

As used herein, the term “target” means any molecule for which thepresence, location and/or concentration is or can be determined.Examples of targets include nucleic acid sequences and proteins, such asthose disclosed herein.

Oligomers and Conjugates

One aspect of the present disclosure is directed to conjugates of aspecific binding entity and an oligomer, i.e. [Specific BindingEntity]-[Oligomer]_(n), wherein n is an integer ranging from 1 to 12. Insome embodiments, the conjugates are PNA conjugates, i.e. a conjugate ofa specific binding entity and an oligomer including a PNA sequence (suchas one having one or more substitutions at a gamma carbon of a PNAbase). In some embodiments, the specific binding entity and the oligomerare coupled via a linker (not illustrated in the above formula), such aslinkers that comprise a cleavable group or moiety. In some embodiments,the oligomer comprises a gamma PNA sequence, an uncharged DNA sequence,or a DNA sequence comprising charged and uncharged bases. In otherembodiments, the oligomer comprises a gamma PNA sequence. In yet otherembodiments, the specific binding entity is an antibody and the oligomerincludes a gamma PNA sequence.

In general, the conjugates disclosed herein are suitable for use inimmunohistochemical assays or in situ hybridization assays, includingmultiplex assays, and thereby may be used as detection probes such thattargets within a tissue sample may be detected. In some embodiments,conjugates can be hybridized to a complimentary PNA, DNA or RNA or likesequence that (i) can itself be directly detected; (ii) carry a specificentity that can be directly detected such as fluorophore, enzyme (HRP);or (iii) which can be indirectly detected, such by carrying a haptenwhich may be recognized by another antibody. The conjugates disclosedherein may also function as molecular “bar codes” which may be used inquantitative analyses.

In some embodiments, a conjugate of the present disclosure has thegeneral structure of Formulas (IA) and (IB):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody, an antibody fragment, a drug/antibody complex, and a nucleicacid;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

Z is selected from the group consisting of a PNA sequence including atleast one nucleotide having a substituent at a gamma position, anuncharged DNA sequence, and a DNA sequence comprising charged anduncharged bases;

X is a label (such as those described herein);

Y is a spacer;

m is 0 or an integer ranging from 1 to 6;

z is 0 or 1; and

n is an integer ranging from 1 to 12.

In some embodiments, Z comprises a DNA sequence having only unchargedbases. In other embodiments, Z comprises a DNA sequence having bothcharged and uncharged bases. In yet other embodiments, Z comprises a DNAsequence wherein at least 50% of the bases of the DNA sequence areuncharged. In yet other embodiments, Z comprises a PNA sequence.

In some embodiments, Z comprises a gamma PNA sequence having at leasttwo gamma PNA bases. In some embodiments, Z comprises a gamma PNAsequence having at least three gamma PNA bases. In some embodiments, Zcomprises a gamma PNA sequence having at least four gamma PNA bases.

In some embodiments, the PNA sequence comprises between about 5 and 60bases. In some embodiments, the PNA sequence comprises between about 5and 30 bases. In some embodiments, the PNA sequence comprises betweenabout 5 and 20 bases. In some embodiments, the PNA sequence comprisesbetween about 5 and 15 bases. In some embodiments, the PNA sequencecomprises between about 5 and 10 bases. In some embodiments, the PNAsequence comprises about 15 bases. In some embodiments, the PNA sequencecomprises about 10 bases.

In some embodiments, the conjugates Formulas (IA) and (IB) include alinker, e.g. a multi-functional linker, designed to couple the specificbinding moiety to the PNA or DNA containing oligomer. In someembodiments, the multi-functional linker is a hetero-bifunctionallinker, i.e. one comprising at least two different reactive functionalgroups (see, for example, the groups A and B defined herein). Forexample, a hetero-bifunctional linker may comprise a carboxylic acidgroup and an amine group, where one of the carboxylic acid group or theamine group is capable of forming a bond with one of the specificbinding entity or the PNA or DNA sequence, and wherein the other of thecarboxylic acid group or amine group is capable of forming a bond withanother of the specific binding entity or the PNA or DNA sequence. Insome embodiments, the “Linker” comprises one or more cleavable groups.In some embodiments, the one or more cleavable groups include aphotocleavable moiety.

In some embodiments ‘Linker’ is a branched or unbranched, linear orcyclic, substituted or unsubstituted, saturated or unsaturated, grouphaving between 2 and 40 carbon atoms, and optionally having one or moreheteroatoms selected from O, N, or S. In other embodiments ‘Linker’ is abranched or unbranched, linear or cyclic, substituted or unsubstituted,saturated or unsaturated, group having between 2 and 20 carbon atoms,and optionally having one or more heteroatoms selected from O, N, or S.

In some embodiments, Y is a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated group havingbetween 1 and 12 carbon atoms and optionally having one or more O, N, orS heteroatoms. In other embodiments, Y is a branched or unbranched,linear or cyclic, substituted or unsubstituted, saturated or unsaturatedgroup having between 1 and 6 carbon atoms and optionally having one ormore O, N, or S heteroatoms. In some embodiments, Y comprises acleavable group, such as those described herein with regard to‘Linkers.’ In some embodiments, n ranges from 1 to 9. In otherembodiments, n ranges from 1 to 6. In some embodiments, X is achromogenic moiety.

In some embodiments, the conjugate is a PNA conjugate having the generalstructure of Formula (IIA):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody, an antibody fragment, a drug/antibody complex, and a nucleicacid;

‘PNA Oligomer’ comprises a PNA sequence including at least onenucleotide having a substituent at a gamma carbon position;

n is an integer ranging from 1 to 12.

In some embodiments, a single PNA oligomer is coupled to a specificbinding entity, e.g. an antibody, a nucleic acid, etc. In otherembodiments, a plurality of PNA oligomers are coupled to a specificbinding entity. In some embodiments, n is an integer ranging from 1 and10. In other embodiments, n is an integer ranging from 1 and 8. In yetother embodiments, n is an integer ranging from 1 and 6. In yet otherembodiments, n is an integer ranging from 2 and 6. In yet otherembodiments, n is an integer ranging from 1 and 4. In yet otherembodiments, n is an integer ranging from 2 and 4. In yet otherembodiments, n is at least 2. In further embodiments, n is at least 3.

The ‘PNA Oligomer’ may include a linker or spacer designed to facilitatethe coupling of a PNA sequence or gamma PNA sequence to the specificbinding entity, as described further herein. In other embodiments, thePNA oligomer may comprise a group which increases the water solubilityof the conjugate, e.g. a linker or spacer that includes functionalitywhich increase the water solubility of the PNA conjugate.

In some embodiments the ‘PNA Oligomer’ includes at least one label toenable direct or indirect detection of the conjugate or a target.

In some embodiments, the PNA conjugates have the structure of Formula(IIB):

wherein

‘Specific binding entity’ is selected from the group consisting of anantibody, an antibody fragment, a drug/antibody complex, and a nucleicacid;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

‘PNA’ is a PNA sequence including at least one nucleotide having asubstituent at a gamma carbon position;

X is a label;

Y is a spacer;

m is 0 or an integer ranging from 1 to 6;

z is 0 or 1; and

n is an integer ranging from 1 to 12.

In some embodiments, Y is a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated group havingbetween 1 and 12 carbon atoms and optionally having one or more O, N, orS heteroatoms. In some embodiments, Y is a branched or unbranched,linear or cyclic, substituted or unsubstituted, saturated or unsaturatedgroup having between 1 and 6 carbon atoms and optionally having one ormore O, N, or S heteroatoms. In some embodiments, Y comprises acleavable group, such as those described herein with regard to‘Linkers.’

In some embodiments, a single PNA oligomer, i.e.-(Linker-PNA-[Y]_(z)-[X]_(m))_(n)), is coupled to a specific bindingentity. In other embodiments, a plurality of PNA oligomers are coupledto a specific binding entity. In some embodiments, n is an integerranging from 1 and 10. In other embodiments, n is an integer rangingfrom 1 and 8. In yet other embodiments, n is an integer ranging from 1and 6. In yet other embodiments, n is an integer ranging from 2 and 6.In yet other embodiments, n is an integer ranging from 1 and 4. In yetother embodiments, n is an integer ranging from 2 and 4. In yet otherembodiments, n is at least 2. In further embodiments, n is at least 3.

In some embodiments, the ‘Specific Binding Entity” is a primary antibodyand the PNA sequence has at least one gamma PNA base, i.e. at least onePNA base which is substituted at the gamma position. In someembodiments, the ‘Specific Binding Entity” is a primary antibody and thePNA sequence has at least two gamma PNA bases. In some embodiments, the‘Specific Binding Entity’ is a primary antibody and the PNA sequence hasat least three gamma PNA bases. In some embodiments, the ‘SpecificBinding Entity’ is a primary antibody and the PNA sequence has at leastfour gamma PNA bases.

In some embodiments, the ‘Specific Binding Entity’ is a primary antibodyand the PNA sequence comprises about 10 bases. In other embodiments, the‘Specific Binding Entity’ is a secondary antibody and the PNA sequencecomprises about 15 bases. In yet other embodiments, the ‘SpecificBinding Entity’ is a primary antibody and the PNA sequence comprisesabout 15 bases, and wherein at least one of the nucleotides in the PNAsequence comprises a substituent at a gamma carbon position. In furtherembodiments, the ‘Specific Binding Entity’ is a primary antibody and thePNA sequence comprises about 15 bases, and wherein at least two of thenucleotides in the PNA sequence comprises a substituent at a gammacarbon position.

In some embodiments, the PNA sequence has at least two gamma PNA bases,i.e. at least two bases having a substituent at a gamma carbon position.In some embodiments, the PNA sequence has at least three gamma PNAbases. In some embodiments, the PNA sequence has at least four gamma PNAbases.

In some embodiments, the PNA sequence comprises between about 5 and 60bases. In some embodiments, the PNA sequence comprises between about 5and 30 bases. In some embodiments, the PNA sequence comprises betweenabout 5 and 20 bases. In some embodiments, the PNA sequence comprisesbetween about 5 and 15 bases. In some embodiments, the PNA sequencecomprises between about 5 and 10 bases. In some embodiments, the PNAsequence comprises about 15 bases. In some embodiments, the PNA sequencecomprises about 10 bases.

In other embodiments, the PNA conjugates of Formula (IIC) have thestructure of Formula (IC):

wherein

‘Ab’ is selected from the group consisting of a primary antibody or asecondary antibody);

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

‘PNA’ is a PNA sequence including at least one nucleotide having asubstituent at a gamma carbon position;

X is a label;

Y is a spacer;

m is 0 or an integer ranging from 1 to 6;

z is 0 or 1; and

n is an integer ranging from 1 to 12.

In some embodiments, Y is a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated group havingbetween 1 and 12 carbon atoms and optionally having one or more O, N, orS heteroatoms. In some embodiments, Y comprises a cleavable group, suchas those described above with regard to ‘Linkers.’

In some embodiments, one or more PNA oligomers, i.e.-(Linker-PNA-[Y]_(z)-[X]_(m))_(n)), are coupled to a primary orsecondary antibody to form a PNA-antibody conjugate. In someembodiments, at least one PNA oligomer is coupled to a primary antibody.In some embodiments, at least one PNA oligomer is coupled to a secondaryantibody. In some embodiments, n is an integer ranging from 1 and 10. Inother embodiments, n is an integer ranging from 1 and 8. In yet otherembodiments, n is an integer ranging from 1 and 6. In yet otherembodiments, n is an integer ranging from 2 and 6. In yet otherembodiments, n is an integer ranging from 1 and 4. In yet otherembodiments, n is an integer ranging from 2 and 4.

The number of PNA oligomers which may be coupled to any particularprimary or secondary antibody depends, of course, on the particularantibody selected and its physical and/or chemical properties. In someembodiments, a degree of labeling of the number of oligomers perantibody ranges from between about 2 and about 10. In other embodiments,the degree of labeling is greater than about 1. In other embodiments,the degree of labeling ranges from about 2 to about 6. In yet otherembodiments, the degree of labeling is about 4. It is believed that arelatively low degree of labeling prevents or mitigates any deleteriouseffects on antibody functionality (e.g. antigen binding or long-termstability of the labeled antibody). Again, it is believed that antibodystability is largely dependent on the antibody itself. Thus, the numberof PNA oligomers per antibody may depend on the ability of antibody totolerate functionalization. Indeed, it is even possible to include PNAoligomers comprising multiple labels (described further herein). Forexample, the PNA oligomer may include one or more fluorophores and/orhaptens. In some embodiments, the one or more haptens may be used fordetection. In some embodiments, the one or more fluorophores may be usedto monitor the binding of the PNA to the antibody.

In some embodiments, the PNA sequence has at least two gamma PNA bases.In some embodiments, the PNA sequence has at least three gamma PNAbases. In some embodiments, the PNA sequence has at least four gamma PNAbases.

In some embodiments, the PNA sequence comprises between about 5 and 60bases. In some embodiments, the PNA sequence comprises between about 5and 30 bases. In some embodiments, the PNA sequence comprises betweenabout 5 and 20 bases. In some embodiments, the PNA sequence comprisesbetween about 5 and 15 bases. In some embodiments, the PNA sequencecomprises between about 5 and 10 bases. In some embodiments, the PNAsequence comprises about 15 bases. In some embodiments, the PNA sequencecomprises about 10 bases.

In some embodiments, a PNA Oligomer has the structure of Formula (III):

wherein

T is a group having a terminal reactive moiety;

‘Linker’ is a branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated, group having between 2 and 80carbon atoms, and optionally having one or more heteroatoms selectedfrom O, N, or S;

‘PNA’ is a PNA sequence including at least one nucleotide having asubstituent at a gamma carbon position;

X is a label;

Y is a spacer;

m is 0 or an integer ranging from 1 to 6; and

z is 0 or 1.

In some embodiments, the PNA sequence has at least two gamma PNA bases.

In some embodiments, the PNA sequence has at least three gamma PNAbases.

In some embodiments, the PNA sequence has at least four gamma PNA bases.

In some embodiments, the PNA sequence has at least one gamma PNA base,and wherein the at least one gamma PNA base is conjugated directly orindirectly to a reporter moiety.

The PNA oligomers may be coupled to any portion of the antibody. Threefunctional groups in antibodies are the sites for covalentmodifications: amines (—NH2), thiol groups (—SH) and carbohydrateresidues (Shrestha D, et al, 2012).

As such, any of the PNA oligomers disclosed herein may be coupled toamine residues, thiol residues, and carbohydrate residues or anycombination thereof.

In some embodiments, the PNA oligomers are coupled to Fc portions of theantibody. In other embodiments, the PNA oligomers are coupled to thehinge regions of the antibody. In some embodiments, the PNA oligomersare coupled to one or more of the Fc regions of the antibody and one ormore of the hinge regions of the antibody. Indeed, any combination iscontemplated by the present disclosure.

Amino groups are generally favored primarily because of the abundance ofthese moieties in the antibody. However, the randomness of amino groupsposes a risk that the antibody may become deactivated. (Adamczyk M, etal, 1999, Bioconjug Chem; Jeanson A, et al, 1988, J Immunol Methods;Vira S, et al, 2010, Anal Biochem; Pearson J E et al, 1998, J ImmunolMethods). In some embodiments, one or more PNA oligomers are coupled toamino groups of an antibody.

On the other hand, and under appropriate reaction conditions, sulfhydryllabeling offers high specificity targeting of the disulfide bondsbetween the two heavy chains of the antibody in the hinge region. Sincethe hinge region is distant from the antigen binding site, thismodification is believed to better preserve antibody's binding affinity.In some embodiments, one or more PNA oligomers are coupled to thiolgroups of an antibody.

Conjugations at the carbohydrate moieties present in the Fc part of theantibody are similar to that of thiol group, such that modificationoccurs at a —CHO group distant from the antigen binding site. Again,without wishing to be bound by any particular theory, it is believedthat conjugation at the carbohydrate offers less of a negative impact onan antibody's binding affinity. The degree of labeling varies dependingon the glycosylation status of a specific antibody. However, loss inantibody affinity was still reported by Jeanson A, et al, 1988, JImmunol Methods. In some embodiments, one or more PNA oligomers arecoupled to carbohydrate groups of an antibody.

In some embodiments, T is a reactive group capable of forming direct abond with a functional group of a specific binding entity, e.g. an aminogroup of an antibody or indirect bond through a linker, e.g. azide onthe PNA sequence bound to the thiol group of the antibody via aDBCO-maleimide bifunctional linker. In some embodiments, T is a NHSester, thiol, maleimide or azide group.

In other embodiments T includes a reactive function group that iscapable of participating in nucleophilic substitutions (e.g., reactionsof amines and alcohols with acyl halides, active esters), electrophilicsubstitutions (e.g., enamine reactions) and additions to carbon-carbonand carbon-heteroatom multiple bonds (e.g., Michael reaction,Diels-Alder addition). These and other useful reactions are discussedin, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley& Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, AcademicPress, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS;Advances in Chemistry Series, Vol. 198, American Chemical Society,Washington, D.C., 1982.

In some embodiments, the reactive functional group is a carboxylic acid,an activated ester of a carboxylic acid, a carbodiimide, a sulfonylhalide, an acyl halide, a silyl halide, an acyl azide, an acyl nitrile,an acrylamide, an amine, an aldehyde, an alkyl halide (wherein thehalide can be later displaced with a nucleophilic group such as, forexample, an amine, a carboxylate anion, thiol anion, carbanion, or analkoxide ion, thereby resulting in the covalent attachment of a newgroup at the site of the halogen atom), an aryl halide, an alkylsulfonate, a sulfonate ester, an anhydride, an azide, an aziridine, adiazoalkane, an haloacetamide, an halotriazine, an hydrazine, anhydroxylamine, an isocyanate, an isothiocyanate, a maleimide, aphosphoramidate, a thiol (which, in some embodiments, can be convertedto disulfides, reacted with acyl halides, or bonded to metals), anhydroxyl (which can be converted to esters, ethers, aldehydes, etc.), anhydrazine and an alkyne (which can undergo, for example, cycloadditions,acylation, Michael addition). In some embodiments, the reactivefunctional group is a carboxyl group or the various derivatives thereofincluding, but not limited to, N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters. Insome embodiments, the reactive functional groups are dienophile groupswhich are capable of participating in Diels-Alder reactions such as, forexample, maleimide groups. In some embodiments, the reactive functionalgroups are aldehyde or ketone groups such that subsequent derivatizationis possible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyl lithium addition.

In other embodiments, the reactive functional group is selected fromcarboxylic acid, an activated ester of a carboxylic acid, sulfonylhalide, acyl halide, amine, alkyl or aryl halide, anhydride, azide,haloacetamide, halotriazine, hydrazine, isocyanate, isothiocyanate,maleimide, phosphoramidate, thiol, hydroxyl and alkyne. In yet otherembodiments, the reactive functional group is selected from carboxylicacid, an activated ester of a carboxylic acid, amine, azide,haloacetamide, hydrazine, isocyanate, maleimide and alkyne.

PNA Sequences

In some embodiments, the PNA sequence is homogenous, i.e. comprising asingle nucleotide type. In other embodiments, the PNA sequence isheterogeneous, i.e. comprising multiple nucleotide types, and thenucleotides may be organized randomly or within repeat groups. In yetother embodiments, the PNA sequence can be designed to encode particularinformation, such as a bar code, as opposed to functioning solely as acarrier.

In some embodiments, the PNA sequence includes from 2 to 60 bases. Inother embodiments, the PNA sequence includes from 2 to 50 bases. In yetother embodiments, the PNA sequence includes from 2 to 40 bases. Infurther embodiments, the PNA sequence includes from 2 to 40 bases. Inyet further embodiments, the PNA sequence includes between 1 and 30bases. In even further embodiments, the PNA sequence includes between 1and 20 bases. In other embodiments, the PNA sequence includes between 20and 40 bases. In yet other embodiments, the PNA sequence includesbetween 20 and 30 bases. In yet other embodiments, the PNA sequenceincludes between 30 and 40 bases. In yet other embodiments, the PNAsequence includes between 5 and 20 bases. In yet other embodiments, thePNA sequence includes between 5 and 15 bases. In yet other embodiments,the PNA sequence includes between 8 and 12 bases. In yet otherembodiments, the PNA sequence includes between 12 and 18 bases. Infurther embodiments, the PNA sequence includes about 10 bases. Infurther embodiments, the PNA sequence includes about 15 bases. Infurther embodiments, the PNA sequence includes at least 10 bases. Infurther embodiments, the PNA sequence includes at least 150 bases.

In some embodiments, one or more PNA nucleotides of any PNA sequence areindependently derivatized or substituted with a moiety on a gamma carbonof a nucleotide (see, for example, FIG. 16 ). It is believed that theT_(m) of gamma PNA (as compared with PNA not having a gamma substituent)is higher by about 5° C. to about 8° C. per single nucleotidesubstitution, which results in more sequence specific binding at higheraffinity. It is also believed that gamma PNA (as compared with PNA nothaving a gamma substituent) can provide several advantages such asimproved solubility, less self-aggregation, more stable PNA-DNA duplexformation, and flexibility for multi labeling and otherfunctionalization. For example, the gamma substituent may be derivatizedwith an amino acid, e.g. lysine, alanine, arginine, glutamic acid, etc.In some embodiments, the one or more charged moieties are lysine orderivatives thereof. In other embodiments, the one or more chargedmoieties are l-(S)-lysine. In yet other embodiments, the one or morecharged moieties are d-(R)-lysine. In further embodiments, the gammasubstituent may be derivatized with thialysine. In some embodiments, theincorporation of lysine facilitates further chemical conjugation.

In some embodiments, one or more PNA nucleotides of any PNA sequence(such as those described herein) are independently derivatized with acharged moiety on a gamma carbon of the PNA base. In some embodiments,one or more of the charged moieties are lysine. In some embodiments, oneor more of the charged moieties are peptides (e.g. a peptide havingbetween 2 and 20 amino acids, a peptide having between 2 and 10 aminoacids, a peptide having between 2 and 8 amino acids, or a peptide havingbetween 1 and 5 amino acids). For example, a peptide may belysine-guanine-lysine or lysine-guanine-guanine-guanine-lysine. By wayof another example, the peptide may be lysine-[U]_(q)-lysine, where Urepresents an amino acid and where q is 0 or an integer ranging from 1to 20. In instances where q is 1 or more, U may represent a homogeneousor heterogeneous short peptide sequence, i.e. one including the same ordifferent amino acids. In some embodiments, U is selected from lysine,alanine, arginine, guanine, glutamic acid or any combination thereof.

In some embodiments, one or more PNA nucleotides of any PNA sequence areindependently substituted at the gamma position with a polymer. In someembodiments, the polymer is one which at least partially imparts thehydrophilicity to the PNA sequence or PNA oligomer. In some embodiments,one or more PNA nucleotides are independently derivatized with ashort-chain oligoethylene moiety on at least one gamma carbon of the PNAsequence.

In some embodiments, one or more PNA nucleotides of any PNA sequence areindependently substituted with a “miniPEG” at a gamma-carbon position.Within the context of the present disclosure, the term “miniPEG” refersto a single poly-ethyleneglycol (PEG) unit or a polymer of PEGcomprising from 2-50 PEG monomers. According to one embodiment, the termminiPEG includes without limitation a —CH₂—(OCH₂—CH₂)_(q)—O—P groupwhere subscript q is an integer between 1-50 and P is selected from thegroup consisting of H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₃-C₈)aryl, (C₃-C₈)cycloalkyl, (C₃-C₈)aryl(C₁-C₆)alkylene and(C₃-C₈)cycloalkyl(C₁-C₆)alkylene. Illustrative of miniPEG units includewithout limitation —CH₂—(OCH₂—CH₂)₁₋₄₅OH, —CH₂—(OCH₂—CH₂)₁₋₄₀OH,—CH₂-(OCH₂—CH₂)₁₋₃₅OH, —CH₂—(OCH₂—CH₂)₁₋₃₀OH, —CH₂—(OCH₂—CH₂)₁₋₂₅OH,—CH₂—(OCH₂—CH₂)₁₋₂₀OH, —CH₂—(OCH₂—CH₂)₁₋₁₅OH, —CH₂—(OCH₂—CH₂)₁₋₁₀OH, and—CH₂—(OCH₂—CH₂)₁₋₅OH groups.

Further illustrative of the class miniPEGs are—CH₂—(OCH₂—CH₂)₁₋₄₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₄₀(C₁-C₈)alkyl,—CH₂—(OCH₂—CH₂)₁₋₃₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₃₀O(C₁-C₈)alkyl,—CH₂—(OCH₂—CH₂)₁₋₂₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₂₀O(C₁-C₈)alkyl,—CH₂—(OCH₂—CH₂)₁₋₁₅O(C₁-C₈)alkyl, —CH₂—(OCH₂—CH₂)₁₋₁₀O(C₁-C₈)alkyl, and—CH₂—(OCH₂—CH₂)₁₋₅O(C₁-C₈)alkyl groups.

Additional examples of gamma substituted PNAs, as well as their methodsof synthesis, are disclosed in United States Patent ApplicationPublication No. 2016/0096867, the disclosure of which is herebyincorporated by reference herein in its entirety.

In some embodiments, the substituent at the gamma position may furthercomprise at least one reporter moiety conjugated directly or indirectlythereto. In some embodiments, the gamma position may be substituteddirectly with a reporter moiety. In other embodiments, the gammaposition may be substituted indirectly with a reporter moiety, such asthrough a short linker group. For example, if the gamma position issubstituted with a lysine residue, a reporter moiety may be conjugatedto the lysine residue.

In some embodiments, any substituent at a gamma carbon position includesmore than one reporter moiety. By way of example only, if the gammaposition is substituted with a miniPEG, a polymer, or a peptide, aplurality of reporter moieties may be conjugated to the miniPEG,polymer, or peptide substituent, accordingly (see, for example, FIG. 17). In some embodiments, a plurality of PNA nucleotides each havesubstituents at the gamma position, providing for a branched PNAoligomer, where each branch includes a miniPEG, a polymer, or a peptidehaving a plurality of reporter moieties. In some embodiments, a gammacarbon of a PNA oligomer may be substituted with another PNA sequence orgamma PNA sequence.

Non-limiting examples of PNA sequences are provided below. In oneembodiment, PNA may have the sequence GTCAACCATCTTCAG (SEQ ID NO: 1). Inanother embodiment, PNA may have the sequence TTAGTCCAACTGGCA (SEQ IDNO: 2). In another embodiment, PNA may have the sequence CATTCAAATCCCCGA(SEQ ID NO: 3). In another embodiment, PNA may have the sequenceCCATCTTCAG (SEQ ID NO: 4). In another embodiment, PNA may have thesequence TTAGTCCAAC (SEQ ID NO: 5). In another embodiment, PNA may havethe sequence CATTCAAATC (SEQ ID NO: 6). In another embodiment, PNA mayhave the sequence CATCCTGCCG (SEQ ID NO: 7). Since the PNA bases are notcharged (like DNA bases) the PNA is believed to be comparatively morehydrophobic than DNA. It is also believed that the PNA hydrophobicity isproportional to the number of bases making that PNA sequence. As such,the more bases a PNA sequence has the higher the hydrophobicity will be.Consequently, a 15 base PNA sequence, for example, is more hydrophobicthan a 10 base PNA sequence. It is also believed that since hydrophobicPNA sequences are not easily dissolved in aqueous solutions they areharder to conjugate to antibodies. Moreover, the conjugation ofhydrophobic PNA sequences on antibodies causes miss-behavior of theconjugate manifested as non-specific binding of the conjugate on thetissue. It is believed that reducing the number of bases of the PNAsequences (for example, from 10 bases from 15 bases) results in higherPNA loading and more stable conjugates.

Any of the SEQ ID NOS 1 through 7 may be modified by those of ordinaryskill the art such that a substituent is present at the gamma positionof any of the PNA bases. Of course, each gamma position may be modifiedwith the same or different substituent (e.g. one base may be substitutedwith a miniPEG having a first molecular weight while another may besubstituted with another miniPEG having a second molecular weight or maybe instead substituted with a lysine). By way of example, any of thesequence listings herein may include one or more substituents at thegamma position, for example GT*CAA*CCA*TCT*TCA*G (SEQ ID NO: 1), where“*” indicates a PNA nucleotide that comprises a substituent at thegamma-carbon position. By way of another example, SEQ ID NO: 2 mayinclude one or more substituents at the gamma position, for exampleTT*AGT*CCA*ACT*GGC*A (SEQ ID NO: 2), where “*” indicates a PNAnucleotide that comprises a substituent at the carbon gamma position. Byway of yet another example, SEQ ID NO: 2 may include one or moresubstituents at the gamma position, for example T*TAGTC*CA*ACT*GGC*A(SEQ ID NO: 2), where “*” indicates a PNA nucleotide that comprises asubstituent at the carbon gamma position. Of course, any of the gammasubstituents may include one or more reporter moieties.

Linkers

In general, and as noted above with regard to Formulas (IA), (IB),(IIA), (IIB), (IIC), and (III), the ‘Linker’ is a branched orunbranched, linear or cyclic, substituted or unsubstituted, saturated orunsaturated, group having between 2 and 80 carbon atoms, and optionallyhaving one or more heteroatoms selected from O, N, or S. In general, theLinker has a molecular weight ranging from about 1 g/mol to about 3000g/mol. In other embodiments, the Linker has a molecular weight rangingfrom about 20 g/mol to about 200 g/mol. In some embodiments, the Linkerhas a length ranging from between about 0.5 nm to about 20 nm. In otherembodiments, the Linker has a length which is less than about 15 nm. Inyet other embodiments, the Linker has a length which is less than about10 nm.

In some embodiments, the ‘Linker’ has the structure depicted in Formula(IVA):

wherein d and e are integers each independently ranging from 2 to 20; Qis a bond, O, S, or N(R^(c))(R^(d)); R^(a) and R^(b) are independentlyH, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d)); R^(c) and R^(d) areindependently CH₃ or H; and A and B are independently a branched orunbranched, linear or cyclic, substituted or unsubstituted, saturated orunsaturated group having between 1 and 12 carbon atoms and optionallyhaving one or more O, N, or S heteroatoms. In some embodiments, d and eare integers ranging from 2 to 6. In some embodiments, and as describedin further detail herein, at least one of A or B comprises a cleavablemoiety.

In some embodiments, the ‘Linker’ has the structure depicted in Formula(IVB):

wherein

d and e are integers each independently ranging from 2 to 20;

Q is a bond, O, S, or N(R^(c))(R^(d));

R^(c) and R^(d) are independently CH₃ or H; and

A and B are independently a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated group havingbetween 1 and 12 carbon atoms and optionally having one or more O, N, orS heteroatoms.

In some embodiments, the “Linker” has the structure depicted in Formula(IVC):

wherein

d and e are integers each independently ranging from 2 to 20;

A and B are independently a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated group havingbetween 1 and 12 carbon atoms and optionally having one or more O, N, orS heteroatoms. In other embodiments, d and e are integers ranging from 2to 15. In other embodiments, d and e are integers ranging from 2 to 10.In yet other embodiments, d and e are integers ranging from 2 to 6.

In some embodiments, the ‘Linker’ comprises solubilizing groups, such aspolyethylene glycol (PEG) groups, to increase the water solubility ofthe PNA conjugates. In some embodiments, the Linkers comprises betweenabout 2 and about 24 PEG groups. In some embodiments, the Linkerscomprises between about 2 and about 18 PEG groups. In other embodiments,the Linkers comprises between about 2 and about 12 PEG groups. In yetother embodiments, the Linkers comprises between about 2 and about 6 PEGgroups. In yet other embodiments, the Linkers comprises 4 PEG groups. Inyet other embodiments, the linker comprises 8 PEG groups. In yet otherembodiments, the Linkers comprises 12 PEG groups. In yet otherembodiments, the Linkers comprises 16 PEG groups. In yet otherembodiments, the Linkers comprises 24 PEG groups. Without wishing to bebound by any particular theory, it is believed that the incorporation ofsuch alkylene oxide linkers is believed to increase the hydrophilicityof the PNA conjugate. A person of ordinary skill in the art willappreciate that as the number alkylene oxide repeat units in the linkerincreases, the hydrophilicity of the PNA conjugate also may increase.Additional heterobifunctional polyalkyleneglycol linkers useful forpracticing certain disclosed embodiments of the present disclosure aredescribed in United States Patent Publication Nos. 2006/0246542 and2009/00181398, the disclosures of each are hereby incorporated byreference herein in their entireties.

In some embodiments, the groups A and B include moieties that arecapable of forming bonds with a group of the Specific Binding Entity ora group of the PNA sequence. In some embodiments, one or both of A or Bis a carbonyl-reactive group. Suitable carbonyl-reactive groups includehydrazine, hydrazine derivatives, and amine. In other embodiments, oneor both of A or B is an amine-reactive group. Suitable amine-reactivegroups include active esters, such as NHS or sulfo-NH S,isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides,aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides,imidoesters, anhydrides and the like. In yet embodiments, one or both ofA or B is a thiol-reactive group. Suitable thiol-reactive groups includenon-polymerizable Michael acceptors, haloacetyl groups (such asiodoacetyl), alkyl halides, SPDP (succinimidyl3-(2-pyridyldithio)propionate), maleimides, aziridines, acryloyl groups,vinyl sulfones, benzoquinones, aromatic groups that can undergonucleophilic substitution such as fluorobenzene groups (such as tetraand pentafluorobenzene groups), and disulfide groups such as pyridyldisulfide groups and thiols activated with Ellman's reagent. Othersuitable reactive groups are described above with regard to the moiety“T.”

In some embodiments, A and/or B include ultraviolet (UV) or visiblelight photocleavable groups. For example, a group may be introduced thatmay be cleaved upon exposure to an electromagnetic radiation sourcehaving a wavelength of between about 200 nm to about 400 nm (UV) orbetween about 400 nm to about 800 nm (visible). In some embodiments, theUV or visible light photocleavable is selected from the group consistingof Arylcarbonylmethyl Groups (including 4-acetyl-2-nitrobenzyl,Dimethylphenacyl (DMP),2-(Alkoxymethyl)-5-methyl-ax-chloroacetophenones, 2,5-DimethylbenzoylOxiranes, and Benzoin groups: 3′,5′-dimethoxybenzoin (DMB)),O-Nitobenzyl Groups (including 1-(2-nitrophenyl)ethyl (NPE),1-(Methoxymethyl)-2-nitrobenzene, 4,5-dimethoxy-2-nitrobenzyl (DMNB);α-carboxynitrobenzyl (α-CNB), o-Nitro-2-phenethyloxycarbonyl Groups,including 1-(2-nitrophenyl)ethyloxycarbonyl and 2-Nitro-2-PhenethylDerivatives, and o-Nitroanilides such as Acylated5-Bromo-7-Nitroindolines); Coumarin-4-ylmethyl Groups (including7-Methoxycoumarin Derivatives); and Arylmethyl Groups (includingo-Hydroxyarylmethyl Groups).

In other embodiments, A and/or B include near-infrared photocleavablegroups. In some embodiments, a group may be introduced that may becleaved upon exposure to an electromagnetic radiation source having awavelength of between about 700 nm to about 1000 nm. Suitablenear-infrared photocleavable groups include cyanine groups, includingC4-dialkylamine-substituted heptamethine cyanines. Without wishing to bebound by any particular theory, it is believed that the incorporation ofa photocleavable linker allows spatial control over the PNA release andultimately a quantitative measurement of the marker expression.

In yet other embodiments, A and/or B include chemically cleavable groupsthat may be cleaved by different chemical reactants, including reducingagents or by induced changes in pH (e.g. cleavage of the group at a pHof less than 7). Suitable chemically cleavable groups includedisulfide-based groups; diazobenzene groups (including 2-(2-alkoxy-4-hydroxy-phenylazo) benzoic acid scaffolds, sensitive to sodiumdithionite); ester bond-based groups (high pH); and acidic sensitivelinkers (such as dialkoxydiphenylsilane linker or acylhydrazone). Avicinal diol cleavable linker may be cleaved by NaIO₄, such as describedin “A simple and effective cleavable linker for chemical proteomicsapplications,” Mol Cell Proteomics, 2013 January; 12(1):237-44. doi:10.1074/mcp.M112.021014. Epub 2012 Oct. 1. In yet further embodiments, Aand/or B include enzymatically cleavable linkers. Suitable enzymaticallycleavable groups include trypsin cleavable groups and V8 proteasecleavable groups.

In some embodiments, the multi-functional linker is selected from onewhich may be orthogonally protected and deprotected, allowing theskilled artisan to conjugate one of the specific binding entity or PNAmoiety at a time to the multi-functional linker, thus preventingunwanted side reactions or side products.

Labels

In some embodiments, X is selected from haptens, fluorophores,chromogens, enzymes, ligands, phosphorescent or chemiluminescent agents,quantum dots, mass spectrometry tags or any other suitable entity. Thetype of label selected depends on the PNA oligomer or PNA conjugatebeing synthesized and the PNA conjugate's ultimate role afterconjugation to an appropriate specific binding entity. For example, insome embodiments labels may be chosen such that when the PNA oligomersare conjugated to an antibody, the labels may be directly detected (e.g.fluoresceins or fluorescein derivatives or analogs). In otherembodiments, labels may be selected such that when the PNA oligomers areconjugated to an antibody, the labels may be indirectly detected (e.g.detection of a hapten label by using a secondary antibody specific forthe hapten, where the secondary antibody is conjugated to a detectablemoiety). Guidance in the choice of labels appropriate for variouspurposes are discussed, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)and Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley-Intersciences (1987), the disclosures ofwhich are incorporated herein by reference.

Fluorophores belong to several common chemical classes includingcoumarins, fluoresceins (or fluorescein derivatives and analogs),rhodamines, resorufins, luminophores and cyanines. Additional examplesof fluorescent molecules can be found in The Handbook—A Guide toFluorescent Probes and Labeling Technologies, Molecular Probes, Eugene,Oreg.

Where the label includes an enzyme a detectable substrate (i.e. asubstrate of the enzyme) such as a chromogenic moiety, a fluorogeniccompound, or a luminogenic compound can be used in combination with theenzyme to generate a detectable signal (a wide variety of such compoundsare commercially available, for example, from Invitrogen Corporation,Eugene Oreg.). Particular examples of chromogenic compounds/substratesinclude diaminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red,bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT),BCIP/NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB),2,2′-azino-di-[3-ethylbenzothiazoline sulphonate](ABTS), o-dianisidine,4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG),o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside(X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blueand tetrazolium violet.

Alternatively, an enzyme can be used in a metallographic detectionscheme. Metallographic detection methods include using an enzyme such asalkaline phosphatase in combination with a water-soluble metal ion and aredox-inactive substrate of the enzyme. In some embodiments, thesubstrate is converted to a redox-active agent by the enzyme, and theredox-active agent reduces the metal ion, causing it to form adetectable precipitate. (see, for example, U.S. patent application Ser.No. 11/015,646, filed Dec. 20, 2004, PCT Publication No. 2005/003777 andU.S. Patent Application Publication No. 2004/0265922; each of which isincorporated by reference herein). Metallographic detection methodsinclude using an oxido-reductase enzyme (such as horseradish peroxidase)along with a water soluble metal ion, an oxidizing agent and a reducingagent, again to for form a detectable precipitate. (See, for example,U.S. Pat. No. 6,670,113, which is incorporated by reference herein).

Examples of haptens are disclosed herein. An example of using massspectrometry to analyze PNA sequences is described in “Peptide nucleicacid characterization by MALDI-TOF mass spectrometry,” Anal Chem. 1996Sep. 15; 68(18):3283-7.

In some embodiments, the X is selected from the group consisting ofdi-nitrophenyl, biotin, digoxigenin, fluorescein, rhodamine, orcombinations thereof. In other embodiments, the X is selected from thegroup consisting of oxazoles, pyrazoles, thiazoles, nitroaryls,benzofurans, triterpenes, ureas, thioureas, rotenoids, coumarins,cyclolignans, or combinations thereof. In yet other embodiments, the Xis selected from the group consisting of 5-nitro-3-pyrazole carbamide,2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid),3-hydroxy-2-quinoxalinecarbamide, 2,1,3-benzoxadiazole-5-carbamide, and2-acetamido-4-methyl-5-thiazolesulfonamide. In yet other embodiments, Xmay be selected from any of the haptens, chromophores, fluorophores, andenzymes which are described further herein (see, e.g. those listed as“reporter moieties” herein).

Other suitable labels are described in PCT Publication WO/2018/002015,the disclosure of which is hereby incorporated by reference herein inits entirety. For example, suitable labels include multi-dye conjugatehaving at least two chromophores coupled directly or indirectly to eachother.

Of course, in some embodiments, the PNA conjugates do not comprise anylabel, i.e. the PNA oligomer portion of the PNA conjugate terminates ina nucleotide.

Synthesis of PNA Conjugates

The PNA conjugates may be synthesized by any means known to know ofordinary skill in the art. In some embodiments, a PNA or gamma PNAoligomer (e.g. one including a linker or a reporter moiety) is coupledto a specific binding entity through a heterobifunctional cross-linker,such as a cross-linker bearing a NHS ester group (e.g. SPDP-PEG₈-NHS) asillustrated in FIG. 1B. Examples of heterobifunctional cross-linkersinclude DBCO-PEGn-maleimide illustrated in FIG. 18 , DBCO-PEGn-NHS,N3-PEGn-NHS, or N3-PEGn-maleimide (where n ranges from 0 to 20).Non-limiting examples of PNA sequences suitable for conjugate includethe following:

PNA 1: (SEQ ID NO: 8) 5′-Biotin-o-GTCAACCATCTTCAG-Lys(C₆SH)-3′ PNA 2:(SEQ ID NO: 9) 5′-Biotin-o-TTAGTCCAACTGGCA-Lys(C₆SH)-3′ PNA 3:(SEQ ID NO: 10) 5′-Biotin-o-CATTCAAATCCCCGA-PL-Lys(C₆SH)-3′ PNA 4:(SEQ ID NO: 11) 5′-Biotin-o-CTGAAGATGGTTTAC-Lys(C₆SH)-3′ PNA 5:(SEQ ID NO: 12) 5′-A1exa488-o-CATCCTGCCGCTATG-Lys(C₆SH)-3′ PNA 6:(SEQ ID NO: 13) 5′-Biotin-o-GTCAACCATCTTCAG-Arg-o-Cys-3′

While the size PNA sequences identified above each have 15 bases, theskilled artisan will appreciate that PNA sequences that are similarlyfunctionalized may comprise any number of bases, e.g. 10 bases. Forexample, the PNA sequence may be: sPNA4: Biotin-o-CCATCTTCAG-Lys(C6SH)(SEQ ID NO: 4).

In some embodiments, a NHS side of a cross-linker is used to bind to anamine group of an antibody to form an amide bond; whereas a(succinimidyl 3-(2-pyridyldithio)propionate) (“SPDP”) side of thecross-linker reacts with a sulfhydryl group at the 3′ end (C terminal)of a PNA sequence forming a disulfide bond. In some embodiments, thedisulfide bond can then be chemically cleaved with a reducing agent.

As described herein, in some embodiments, the cleaved PNA sequence maybe detected and measured with the NanoString nCounter platform. Forexample, the PNA sequence may include a biotin on the 5′ end (Nterminal) to enable direct immobilization on the streptavidin surfacewithout the need of a capture strand (FIG. 1B). This modification isexpected to enable the use of much shorter PNA sequence (about 15 bases)to be hybridized directly to the reporter strand and further analyzed bythe nCounter platform. In some embodiments, a PNA sequence having about15 bases provides enough binding affinity and specificity to thereporter sequence during the detection. Without wishing to be bound byany particular theory, it is believed that the Tm of a 15-mer PNA-DNAcomplex is similar to 50-mer DNA-DNA complex. Examples of suitable PNAsequences for coupling are provided below:

In some embodiments, a photocleavable (PC) linker can be incorporatedbetween the PNA sequence and the specific binding entity (e.g. anantibody) (see the groups identified as A or B defined herein) to allowlight-triggered release of the PNA. In some embodiments, the PNAsequence can be synthesized with the photocleavable linker: Biotin-o-CATTCA AAT CCC CGA-PL-Lys(C6SH) (SEQ ID NO: 10). Following lightirradiation, the PNA sequence may be released intact and may be measuredusing any of the techniques described herein. Alternatively, aphotocleavable bifunctional linker can be synthesized (FIG. 10 ) andused to link the PNA to the antibody.

Multiple PNA oligomers (having either the same or different sequences)can be conjugated to an antibody via different linkers. Such PNAoligomers may have the same or different cleavable groups (e.g. the PNAsequence may be the same but the linker incorporating the cleavablegroup may be different). Some PNA oligomers may be cleavable whileothers are not; or some might be cleavable under certain conditionswhile others are not cleavable under the same conditions.

The approach of incorporating the photocleavable liker within the PNAduring the synthesis of the PNA conjugate is advantageous as it is moretime and cost effective and ensures that the photocleavable linker isincorporated in all the PNA oligomers. Alternatively, a photocleavablebifunctional linker can be synthesized (FIG. 10 ) and used to link PNAsequences to an antibody. This approach is advantageous as thephotocleavable bifunctional linker may be used to link any PNA sequenceto an antibody even if the PNA was not designed to be photocleavable.Without wishing to be bound by any particular theory, this approach mayprovide some flexibility in terms of using the same PNA sequence asnormal (not photocleavable) and photocleavable antibody tag. In someembodiments, the disulfide bond is still present in the synthesizedphotocleavable PNA allowing both light and chemical cleavage. In someembodiments, the biotin moiety is also retained to allow SA-HRP and DABdetection on slides, as noted further herein (see, e.g., FIG. 15 ).

In some embodiments, a PNA sequence may be introduced to the specificbinding moiety and/or linker using “click chemistry,” such asillustrated in FIG. 18 . A non-limiting example of a PNA sequence havinga reactive group (e.g. an azide) capable of undergoing a “click”reaction is presented below:

PNA 7: (SEQ ID NO: 14) 5′-Biotin-O-GTCAACCATCTTCAG-Lys(eg3-N3)-3′

The skilled artisan will appreciate that a PNA sequence comprising anappropriate reactive group may form a click adduct with another moleculethat is also appropriately functionalized to undergo the “click”reaction. Indeed, the skilled artisan will recognize that for one memberof a pair of click conjugates to react with another member of the pairof click conjugates, and thus form a covalent bond, the two members ofthe pair of click conjugates must have reactive functional groupscapable of reacting with each other. The table which follows exemplifiesdifferent pairs of reactive functional groups that will react with eachother to form a covalent bond.

Reactive Functional Group on a Reactive Functional Group on a FirstMember of a Pair of Click Second Member of a Pair of Click ConjugatesConjugates DBCO Azide Alkene Tetrazine TCO Tetrazine Maleimide ThiolDBCO 1,3-Nitrone Aldehyde or ketone Hydrazine Aldehyde or ketoneHydroxylamine Azide DBCO Tetrazine TCO Thiol Maleimide 1,3-Nitrone DBCOHydrazine Aldehyde or ketone Hydroxylamine Aldehyde or ketone TetrazineAlkene

In some embodiments, groups present on an antibody (e.g. a primary orsecondary antibody) are reduced in the presence of dithiothreitol(“DTT”) so as to provide an antibody having one or more thiol groups.The thiol groups may be reacted with a first member of a pair of clickmembers, the first member bearing a reactive functional group capable ofparticipating in the “click chemistry” reaction (e.g. a DBCO group). Thefirst member of the pair of click conjugates may also comprise a secondfunctional group capable of reacting with the thiolated antibody (e.g. amaleimide). The second functional group on the first member of the pairof click conjugates is one which is not capable of reacting in the clickchemistry reaction. As illustrated in FIG. 18 , this step allows theantibody to become functionalized with a first reactive functional groupcapable of participating in the “click chemistry” coupling. A secondmember of the pair of click members is then introduced, such as a PNAmolecule including a second reactive functional group capable ofparticipating in the “click chemistry” reaction (e.g. an azide group).The second member of the pair of click members may also comprise a labelor reporter moiety. In the embodiment depicted in FIG. 18 , the DBCOgroup-bearing antibody is able to couple to with the azide group of thesecond member of the pair of click members, such that the PNA conjugatebecomes coupled to the antibody. The PNA conjugated in this procedure isnot chemical cleavable or photo-cleavable

In some embodiments, a PNA sequence may be introduced to the specificbinding moiety and/or linker using “maleimide” chemistry (see FIG. 20 ).In some embodiments, the PNA conjugated in this procedure is notchemical cleavable or photo-cleavable

A non-limiting example of a PNA sequence having a SMCC group ispresented below:

PNA 8: (SEQ ID NO: 15) 5′-Biotin-O-GTCAACCATCTTCAG-Lys(SMCC)-3′

Without wishing to be bound by any particular theory, it is believedthat the use of click chemistry or maleimide chemistry permits theintroduction of shorter PNA sequences, e.g. sequences having 10 or lessbases.

Detection of Conjugates

In some embodiments, the conjugate of any of Formulas (IA), (IB), (IIA),(IIB), and (IIC), may comprise a label that facilitates the directdetection of the conjugate. For example, if the label of the conjugatecomprises a fluorophore or a chromophore, the fluorophore or chromophoremay be directly detected according to methods known to those of ordinaryskill in the art.

In other embodiments, specific reagents are utilized to enable detectionof any the conjugates of Formulas (IA), (IB), (IIA), (IIB), and (IIC),and hence the targets in a tissue sample. In some embodiments, detectionreagents are utilized which are specific to the particular label of theconjugate or that are complementary to the nucleotide sequence (e.g. aPNA sequence) of the conjugate, as noted further herein. In someembodiments, the detection reagents comprise a secondary antibody whichis specific for the label of the conjugate. For example, the secondaryantibody may be an anti-label antibody including a label (i.e. ‘X’ inFormulas (IA), (IB), (IIA), (IIB), and (IIC) herein). In someembodiments, the secondary antibody is an anti-hapten antibody and thelabel is a hapten.

In some embodiments, the secondary antibody or anti-label antibody maybe conjugated to a “reporter moiety” to effectuate detection of theconjugate of Formulas (I), (IA), (IC), (ID), and (II). In someembodiments, the reporter moiety of the secondary antibody includeschromogenic, fluorescent, phosphorescent and luminescent molecules andmaterials, catalysts (such as enzymes) that convert one substance intoanother substance to provide a detectable difference (such as byconverting a colorless substance into a colored substance or vice versa,or by producing a precipitate or increasing sample turbidity), haptensthat can be detected through antibody-hapten binding interactions usingadditional detectably labeled antibody conjugates, and paramagnetic andmagnetic molecules or materials. Of course, the reporter moieties canthemselves also be detected indirectly, e.g. if the reporter moiety is ahapten, then yet another antibody specific to that reporter moiety maybe utilized in the detection of the reporter moiety, as known to thoseof ordinary skill in the art.

In some embodiments, the anti-label antibody includes a reporter moietyselected from the group consisting of DAB; AEC; CN; BCIP/NBT; fast red;fast blue; fuchsin; NBT; ALK GOLD; Cascade Blue acetyl azide;Dapoxylsulfonic acid/carboxylic acid succinimidyl ester; DY-405; AlexaFluor 405 succinimidyl ester; Cascade Yellow succinimidyl ester;pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue succinimidylester; DY-415; 7-hydroxycoumarin-3-carboxylic acid succinimidyl ester;DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; iodoacetamide; AlexaFluor 430 succinimidyl ester; Dabcyl succinimidyl ester; NBDchloride/fluoride; QSY 35 succinimidyl ester; DY-485XL; Cy2 succinimidylester; DY-490; Oregon Green 488 carboxylic acid succinimidyl ester;Alexa Fluor 488 succinimidyl ester; BODIPY 493/503 C3 succinimidylester; DY-480XL; BODIPY FL C3 succinimidyl ester; BODIPY FL C5succinimidyl ester; BODIPY FL-X succinimidyl ester; DYQ-505; OregonGreen 514 carboxylic acid succinimidyl ester; DY-510XL; DY-481XL;6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidyl ester(JOE); DY-520XL; DY-521XL; BODIPY R6G C3 succinimidyl ester; erythrosinisothiocyanate; 5- carboxy-2′,4′,5′,7′-tetrabromosulfonefluoresceinsuccinimidyl ester; Alexa Fluor 532 succinimidyl ester;6-carboxy-2′,4,4′,5′7,7′-hexachlorofluorescein succinimidyl ester (HEX);BODIPY 530/550 C3 succinimidyl ester; DY-530; BODIPY TMR-X succinimidylester; DY-555; DYQ-1; DY-556; Cy3 succinimidyl ester; DY-547; DY-549;DY-550; Alexa Fluor 555 succinimidyl ester; Alexa Fluor 546 succinimidylester; DY-548; BODIPY 558/568 C3 succinimidyl ester; Rhodamine red-Xsuccinimidyl ester; QSY 7 succinimidyl ester; BODIPY 564/570 C3succinimidyl ester; BODIPY 576/589 C3 succinimidyl ester;carboxy-X-rhodamine (ROX); succinimidyl ester; Alexa Fluor 568succinimidyl ester; DY-590; BODIPY 581/591 C3 succinimidyl ester;DY-591; BODIPY TR-X succinimidyl ester; Alexa Fluor 594 succinimidylester; DY-594; carboxynaphthofluorescein succinimidyl ester; DY-605;DY-610; Alexa Fluor 610 succinimidyl ester; DY-615; BODIPY 630/650-Xsuccinimidyl ester; erioglaucine; Alexa Fluor 633 succinimidyl ester;Alexa Fluor 635 succinimidyl ester; DY-634; DY-630; DY-631; DY-632;DY-633; DYQ-2; DY-636; BODIPY 650/665-X succinimidyl ester; DY-635; Cy5succinimidyl ester; Alexa Fluor 647 succinimidyl ester; DY-647; DY-648;DY-650; DY-654; DY-652; DY-649; DY-651; DYQ-660; DYQ-661; Alexa Fluor660 succinimidyl ester; Cy5.5 succinimidyl ester; DY-677; DY-675;DY-676; DY-678; Alexa Fluor 680 succinimidyl ester; DY-679; DY-680;DY-682; DY-681; DYQ-3; DYQ-700; Alexa Fluor 700 succinimidyl ester;DY-703; DY-701; DY-704; DY-700; DY-730; DY-731; DY-732; DY-734; DY-750;Cy7 succinimidyl ester; DY-749; DYQ-4; and Cy7.5 succinimidyl ester.

Fluorophores belong to several common chemical classes includingcoumarins, fluoresceins (or fluorescein derivatives and analogs),rhodamines, resorufins, luminophores and cyanines. Additional examplesof fluorescent molecules can be found in Molecular Probes Handbook—AGuide to Fluorescent Probes and Labeling Technologies, Molecular Probes,Eugene, Oreg., Thermofisher Scientific, 11th Edition. In otherembodiments, the fluorophore is selected from xanthene derivatives,cyanine derivatives, squaraine derivatives, naphthalene derivatives,coumarin derivatives, oxadiazole derivatives, anthracene derivatives,pyrene derivatives, oxazine derivatives, acridine derivatives,arylmethine derivatives, and tetrapyrrole derivatives. In otherembodiments, the fluorescent moiety is selected from a CF dye (availablefrom Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY(available from Invitrogen), Alexa Fluor (available from Invitrogen),DyLight Fluor (e.g. DyLight 649) (available from Thermo Scientific,Pierce), Atto and Tracy (available from Sigma Aldrich), FluoProbes(available from Interchim), Abberior Dyes (available from Abberior), DYand MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (availablefrom Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau andSquare Dyes (available from SETA BioMedicals), Quasar and Cal Fluor dyes(available from Biosearch Technologies), SureLight Dyes (available fromAPC, RPEPerCP, Phycobilisomes)(Columbia Biosciences), and APC, APCXL,RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

In other embodiments, the anti-label antibody is conjugated to anenzyme. In some embodiments, suitable enzymes include, but are notlimited to, horseradish peroxidase, alkaline phosphatase, acidphosphatase, glucose oxidase, β-galactosidase, β-glucuronidase orβ-lactamase. In other embodiments, enzymes include oxidoreductases orperoxidases (e.g. HRP, AP). In these embodiments, the enzyme conjugatedto the anti-label antibody catalyzes conversion of a chromogenicsubstrate to a reactive moiety which covalently binds to a sampleproximal to or directly on the target. Particular non-limiting examplesof chromogenic compounds/substrates include diaminobenzidine (DAB),4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate(BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, APblue, tetramethylbenzidine (TMB), 2,2′-azino-di-[3-ethylbenzothiazolinesulphonate](ABTS), o-dianisidine, 4-chloronaphthol (4-CN),nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD),5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal),methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue,tetrazolium violet,N,N′-biscarboxypentyl-5,5′-disulfonato-indo-dicarbocyanine (Cy5),4-(dimethylamino) azobenzene-4′-sulfonamide (DABSYL),tetramethylrhodamine (DISCO Purple), and Rhodamine 110 (Rhodamine). DAB,which is oxidized in the presence of peroxidase and hydrogen peroxide,results in the deposition of a brown, alcohol-insoluble precipitate atthe site of enzymatic activity.

In some embodiments, the chromogenic substrates are signaling conjugateswhich comprise a latent reactive moiety and a chromogenic moiety. Insome embodiments, the latent reactive moiety of the signaling conjugateis configured to undergo catalytic activation to form a reactive speciesthat can covalently bond with the sample or to other detectioncomponents. The catalytic activation is driven by one or more enzymes(e.g., oxidoreductase enzymes and peroxidase enzymes, like horseradishperoxidase) and results in the formation of a reactive species. Thesereactive species are capable of reacting with the chromogenic moietyproximal to their generation, i.e. near the enzyme. Specific examples ofsignaling conjugates are disclosed in US Patent Publication No.2013/0260379, the disclosure of which is hereby incorporated byreference herein in its entirety.

In embodiments where the label is biotin, the PNA conjugate may becontacted with streptavidin coupled to an enzyme (e.g. alkalinephosphatase or horseradish peroxidase). Without wishing to be bound byany particular theory, it is believed that the streptavidin-AP orstreptavidin-HRP conjugates bind specifically and irreversibly to thebiotin-labeled PNA conjugates. The PNA-biotin-streptavidin conjugatesmay then be visualized using a chromogenic substrate for alkalinephosphatase or horseradish peroxidase (such as those noted above) toproduce a detectable signal. Likewise, where the label is biotin, thePNA conjugate may alternatively be contacted with streptavidin coupledto a fluorophore (e.g. FTIC) and the signals for the fluorophoredetected.

In embodiments where the PNA tag is cleaved from the antibody conjugates(via a cleavable linker as noted herein), the PNA sequence cleaved fromthe conjugate may be detected according to the methods described in“Peptide nucleic acid characterization by MALDI-TOF mass spectrometry,”Anal Chem. 1996 Sep. 15; 68(18):3283-7, the disclosure of which areincorporated by reference herein in their entirety. Likewise, the PNAsequence of the PNA conjugate may similarly be detected by other massspectrometry such as electrospray ionization (ESI) according methodsknown to those of ordinary skill in the art.

Detection of Conjugates Using Complementary Nucleotide Sequences,Including Complementary PNA or DNA Sequences

In some embodiments, conjugates of Formulas (IA), (IB), (IIA), (JIB),and (IIC) may be detected by hybridizing one of a PNA sequence, gammaPNA sequence or a DNA sequence to a nucleotide sequence of theconjugate, the PNA sequence or DNA sequence being complementary to thenucleotide sequence of the conjugate. For example, a PNA or DNA sequencecomplementary to a PNA sequence of a PNA conjugate may be detectedfollowing its hybridization to the PNA sequence of the PNA conjugate.

In some embodiments, the conjugate of Formulas (IA), (IB), (IIA), (IIB),and (IIC) do not comprise a label. In some embodiments, PNA or DNAsequences complementary to the nucleotide sequence of the conjugatecomprises a reporter moiety, such as those described herein. In someembodiments, the reporter moiety is a chromogen. In other embodiments,the reporter moiety is a fluorophore (see, for example, FIG. 8E). In yetother embodiments, the reporter moiety is a hapten (e.g. digoxigenin,such as in FIG. 9D). In further embodiments, the reporter moiety is anenzyme. In even further embodiments, the reporter moiety is ananoparticle (e.g. a gold nanoparticle, which may be used in scanningelectronic imaging or a quantum dot). Of course, in the case of gammaPNA sequences, multiple reporter moieties may be incorporated into theconjugate, such as described herein and illustrated in FIG. 17 .

Of course, the skilled artisan will appreciate that the same conjugateof Formulas (IA), (IB), (IIA), (IIB), and (IIC) may be used to providemultiple imaging modalities. For example, if a fluorescently labeledcomplimentary DNA or PNA sequence is provided, it can be used forfluorescence imaging. With the same conjugate of Formulas (IA), (IB),(IIA), (IIB), and (IIC), a haptenated DNA or PNA sequence can also beused for effect chromogenic imaging.

Detection and/or Quantification of Conjugates Using a NanostringNCounter Platform In some embodiments, the conjugates of Formulas (IA),(IB), (IIA), (JIB), and (IIC)comprise an oligomer having a nucleotidesequence that may serve as a molecular “bar code.” For example, whiletwo PNA conjugates may comprise similar PNA oligomer portions, the PNAoligomer portions may differ in certain bases within the PNA sequence(e.g. even a single change of one nucleotide). In this manner, PNAconjugates having different PNA sequences may be detected and/orquantified, such as by using the Nanostring nCounter Platform. In someembodiments, the PNA conjugates comprise a reporter moiety, such as abiotin label.

In some embodiments, the conjugates of any of Formulas (IA), (IB),(IIA), (IIB), and (IIC) comprise a cleavable linker. Following theintroduction of the conjugate of any of Formulas (IA), (IB), (IIA),(IIB), and (IIC)) to the sample, chemical reagents, enzymes, and/orradiation (e.g. UV, IR, etc.) are introduced to the sample to cleave agroup of the cleavable linker, thus releasing the nucleotide sequence(e.g. a PNA-antibody sequence or a gamma PNA sequence) of the conjugate.This may, of course, be repeated for different conjugates of any ofFormulas (IA), (IB), (IIA), (IIB), and (IIC). Once all of the nucleotidesequences (e.g. PNA sequences or gamma PNA sequences) have beenreleased, they may be detected and quantified as noted herein. Theskilled artisan will also appreciate that different conjugates maycomprise different cleavable linkers, and thus the different nucleotidesequences may be released at different times following introduction ofdifferent reagents/radiation, thus allowing step-wise detection and/orquantification. In some embodiments, the conjugates of any of Formulas(IA), (IB), (IIA), (IIB), and (IIC) are PNA conjugates, i.e. theconjugates comprise an oligomer including a PNA sequence or a gamma PNAsequence.

By way of example, where PD-L1 and Ki67 markers both exist on the sametissue section, an anti-PD-L1/PNA1 conjugate and anti-Ki67 PNA 2conjugate may be used to stain the tissue section. The PNA1 and PNA2oligomer portions may comprise have two different PNA sequences, yetboth are chemically cleavable by means of a cleavable moiety within theconjugate, as noted herein. After incubating the tissue with the twoconjugated antibodies and careful rinsing to remove unboundPNA-conjugated antibodies, the two different PNAs are cleaved from theconjugate. The two different PNAs can be counted on nCounter (NanoStringTechnology) and the number of cleaved PNAs determined. The differencebetween the number of PNA1 and PNA2 reflect the difference in the levelof protein expression of the PD-L1 and Ki67 markers.

In these embodiments, the PNA sequences of the PNA conjugates may bedetected and/or quantified in a similar manner to DNA since thedetection scheme is based the hybridization of a reporter strand to thetarget oligomer (can be DNA or PNA). The PNA conjugates, however, areshorter than the standard DNA targets typically detected by theNanoString nCounter platform (which are about 70-100 bases in length).However, the presence of the biotin on the 3′ end of the PNA excludesthe need to use a capture strand. Moreover, the higher binding affinityof PNA to DNA compared to DNA to DNA provides enough stability to therelatively short PNA/DNA reporter duplex. The PNA sequence will be mixedwith the reporter strand that is designed to be the complement to thePNA sequence. After removing unbound PNA the PNA/reporter constructs areincubated on the streptavidin-coated cartridge then aligned usingelectric field. The nCounter will be used to read and count the reporterstrand. The same procedure can be done with multiple PNA sequences.

Detection and/or Quantification of Conjugates Using Gyros

Gyros is an immunoassay platform using an affinity flow-through formatwith parallel processing and laser-induced fluorescence detection. Theassay is performed in a compact disk (CD) containing over 100nanoliter-scale channels which uses centrifugal force and capillaryaction for liquid delivery and movement.

In a typical non-limiting experiment, a biotinylated epitope peptide isfirst bound to a 15 nL affinity capture column consisting ofstreptavidin-coated beads. After rinsing, a primary antibody specific tothe epitope peptide flows through the column and binds to the peptide. Afluorescent dye (e.g. Alexa Fluor 647) labeled secondary antibody thenbinds to the primary antibody which is then detected and quantifiedusing laser-induced fluorescence. For the detection of the oligomers ofthe present disclosure, the biotinylated PNA oligomer (see, e.g. FIG.3D) is hybridized to a complementary single stranded DNA, which isconjugated to a reporter moiety (e.g. a hapten, including but notlimited to digoxigenin). The biotin in the hybridized nucleic acidstrands binds to the streptavidin-coated beads in the Gyros CD. The DIGlabel on the other end of the hybrid is then detected by Ms-anti-DIGantibody followed by Alexa Fluor 647 labeled goat-anti-mouse antibody(GAM), which facilitates quantitative measurement of the originaloligomer. The principle of using Gyros technology for quantification isillustrated in FIG. 32 . Additional information regarding GyrosTechnology devices and their methods of use are described in U.S. Pat.Nos. 8,133,438 and 8,592,219, the disclosures of which are incorporatedby reference herein in their entireties. Additional informationpertaining to Gyros Technology devices and their methods of use are alsodescribed in US Patent Application Publication Nos. 2011/0116972,2011/0195524, and 2007/0241061, the disclosures of which are herebyincorporated by reference herein in their entireties.

Any conjugate of Formulas (IA), (IB), (IIA), (IIB), and (IIC) may beused for quantification using the Gyros platform, provided that theconjugate includes a linker capable of being cleaved (e.g. a linkerincluding a disulfide group). Following the introduction of theconjugate of any of Formulas (IA), (IB), (IIA), (IIB), and (IIC) to thesample, chemical reagents, enzymes, and/or radiation are introduced tothe sample to cleave a group of the cleavable linker, thus releasing thenucleotide sequence (e.g. a PNA sequence or a gamma PNA sequence) of theconjugate. Quantification may then proceed as noted above.

In some embodiments, the conjugates of any of Formulas (IA), (IB),(IIA), (IIB), and (IIC) are PNA conjugates, i.e. the conjugates comprisean oligomer including a PNA sequence or a gamma PNA sequence, the PNAconjugate comprising a primary antibody conjugated to a PNA oligomer asdescribed herein. In some embodiments, the introduced single strandedDNA is complementary to and capable of hybridizing with the PNA sequenceof the PNA conjugate. In some embodiments, the complementary singlestranded DNA sequence is conjugated to a reporter moiety. In someembodiments, the complementary single stranded DNA sequence isconjugated to a hapten. In some embodiments, the complementary singlestranded DNA sequence is conjugated to digoxigenin.

In some embodiments, multiple, different PNA conjugates may beintroduced simultaneously or sequentially. The skilled artisan will alsoappreciate that the different PNA conjugates may comprise differentcleavable moieties, and thus the different PNA sequences may be releasedat different times following introduction of differentreagents/radiation, thus allowing step-wise quantification.

In some embodiments, following quantification with the Gyros platform,the tissue is stained according to methods common used in the art. Forexample, following cleavage of a PNA or gamma PNA sequence from the PNAconjugate, the tissue in which the PNA conjugate was bound to may be maybe stained by introducing an anti-primary antibody including a reportermoiety, such as illustrated in FIG. 33 . In this way, quantification canbe combined with visualization of a target in a biological sample.

Detection Kits Comprising PNA Conjugates and Detection Reagents forDetecting PNA Conjugates

In some embodiments, the conjugates of Formulas (IA), (IB), (IIA),(IIB), and (IIC), may be utilized as part of a “detection kit.” Ingeneral, any detection kit may include one or more conjugates ofFormulas (IA), (IB), (IIA), (JIB), and (IIC), and detection reagents fordetecting the one or more conjugates.

In some embodiments, the detection kits may include a first compositioncomprising a conjugate of any of Formulas (IA), (IB), (IIA), (JIB), and(IIC), and a second composition comprising detection reagents specificto the first composition, such that the conjugate may be detected viathe detection kit. In some embodiments, the detection kit includes aplurality of conjugates of Formulas (IA), (IB), (IIA), (IIB), and (IIC),(such as those mixed together in a buffer, or those that are provided inindividual shipping containers or compartments), where the detection kitalso includes detection reagents specific for each of the plurality ofconjugates.

By way of example, a kit may include a first PNA conjugate specific fora first target, the PNA conjugate having a first PNA oligomer portion,and a second PNA conjugate specific for a second target having a secondPNA oligomer portion, wherein at least a portion of the first and secondPNA oligomers are different. The kit may further comprise detectionreagents specific for each of the different PNA conjugates.

By way of another example, a kit may include a first PNA conjugatehaving a first PNA oligomer portion (and one that does not comprise alabel) and the kit may further include a PNA or DNA sequence that iscomplementary to the PNA sequence of the first PNA oligomer portion.

By way of yet another example, a kit may include a series of differentPNA conjugates, each PNA conjugate specific to a different target andhaving a different PNA oligomer portion. Each different PNA conjugate ofthe kit may serve as a different molecular “bar code,” which could beused in a qualitative and/or a quantitative analysis.

Of course, any kit may include other agents, including buffers;counterstaining agents; enzyme inactivation compositions;deparaffinization solutions, etc. as needed for manual or automatedtarget detection. The detection kits may also comprise other specificbinding entities (e.g. nucleic acid probes for ISH; unmodified (native)antibodies, and antibody conjugates) and detection reagents to detectthose other specific binding entities. For example, a kit may compriseone or more PNA conjugates; one or more anti-label antibodies fordetecting the one or more PNA conjugates; at least one unmodifiedantibody (i.e. native antibody not coupled to a PNA sequence); anddetection reagents for detecting the at least one unmodified antibody.In some embodiments, instructions are provided for using the PNAconjugates, and other components of the kit, for use in an assay, e.g. aMIHC assay.

Methods of Detecting Targets with the Conjugates of any of Formulas(Ia), (Ib), (IIa), (IIb), and (IIc), and Detection Reagents

The present disclosure also provides methods of detecting one or moretargets within a tissue sample using any of the conjugates of Formulas(IA), (IB), (IIA), (IIB), and (IIC) described herein. In someembodiments, a conjugate of any of Formulas (IA), (IB), (IIA), (IIB),and (IIC) may be used in a simplex assay to directly or indirectlydetect a particular target within the tissue sample (e.g. CD68, Ki67,CD20, etc.).

In some embodiments, the conjugates of any of Formulas (IA), (IB),(IIA), (IIB), and (IIC) comprise a primary antibody (e.g. an antibodyspecific to CD68, Ki67, CD20, etc.). In these embodiments, theconjugates comprising a primary antibody may be used to directly “label”a target with a conjugate. In other embodiments, the conjugates of anyof Formulas (IA), (IB), (IIA), (JIB), and (IIC) comprise a secondaryantibody. In these embodiments, and as discussed in more detail herein,a target (e.g. a protein target or a nucleic acid target) may be labeledwith a primary antibody (for IHC) or a nucleic acid conjugate (e.g. anucleic acid sequence coupled to a hapten, for ISH), and then theprimary antibody or the nucleic acid conjugate may subsequently be“labeled” with a conjugate comprising a secondary antibody. These andother embodiments are described further herein.

In some embodiments, the PNA conjugates comprise a primary antibodywhere the PNA-primary antibody conjugate is specific to a target ofinterest, and where upon application of the PNA-primary antibodyconjugate to the tissue sample, a target-PNA-primary antibody conjugatecomplex is formed (see, for example, FIGS. 22A-22D, and FIG. 26 ).Following application of the PNA-primary antibody conjugate, detectionreagents (e.g. an anti-label antibody) may subsequently be applied suchthat the target-PNA-primary antibody conjugate complex may be detected.In some embodiments, the detection reagents comprise an anti-labelantibody specific to the particular label of the PNA-primary antibodyconjugate, where the anti-label antibody comprises a reporter moiety.The single target may then be visualized or otherwise detected.

In other embodiments, a tissue sample is first contacted with a primaryantibody or a nucleic acid probe, forming either a target-primaryantibody complex or a target-nucleic acid probe complex. Subsequently, aPNA conjugate comprising a secondary antibody is introduced to thetissue sample, the secondary antibody portion of the PNA conjugate beingspecific to either the (i) primary antibody, (ii) a label conjugated tothe primary antibody, or (iii) a label conjugated to the nucleic acidprobe. Application of the PNA-secondary antibody conjugate allowsformation of a secondary complex, allowing the target to be “labeled.”Following application of the PNA-secondary antibody conjugate andformation of the secondary complex, detection reagents (e.g. ananti-label antibody) may be applied such that the secondary complex maybe detected. In some embodiments, the detection reagents comprise ananti-label antibody specific to the particular label of thePNA-secondary antibody conjugate, where the anti-label antibodycomprises a reporter moiety. The target may then be visualized orotherwise detected.

In yet other embodiments, a tissue sample is first contacted with aPNA-primary antibody conjugate; or first contacted with a primaryantibody followed by the introduction of a PNA-secondary antibodyconjugate. Following introduction of the respective PNA conjugate, thesample may be contacted with a DNA or PNA sequence that is complementaryto the PNA sequence of the PNA conjugate, the complementary DNA or PNAsequence comprising one or more reporter moieties (e.g. a chromogen, afluorophore, an enzyme, or a hapten). In embodiments where thecomplimentary DNA or PNA sequence comprises a chromogen or afluorophore, the “labeled” target complex may be directly detected. Onthe other hand, where the complementary DNA or PNA sequence comprises ahapten, an anti-hapten antibody conjugated to a reporter moiety must beintroduced to facilitate eventual detection of the “labeled” targetcomplex.

Of course, and as an alternative embodiment, DNA or PNA sequences of theconjugates of any of Formulas (IA), (IB), (IIA), (IIB), and (IIC) may bequantified such as with the NanoString nCounter method or Gyrostechnology described herein, following cleavage of the DNA or PNAsequence from the conjugate. In some embodiments, the conjugatescomprise a cleavable group and a biotin label. These methods would notrequire the use of any further detection reagents.

In some aspects of the present disclosure are provided methods ofmultiplex detection, including automated multiplex detection. FIG. 14Aprovides a flowchart illustrating one method for the multiplex detectionof targets where a tissue sample is contacted simultaneously with aplurality of PNA conjugates (step 100), where each PNA conjugate isspecific for a particular target, and where each PNA conjugate comprisesa different PNA oligomer (i.e. a PNA oligomer having a different PNAsequence and/or a different label). While FIG. 14A depicts theapplication of PNA conjugates, the skilled artisan will understand thatPNA conjugates may comprise PNA-nucleic acid conjugates, PNA-primaryantibody conjugates, and PNA-secondary antibody conjugates, depending onthe target within the sample (e.g. a nucleic acid sequence, a proteintarget recognized by a primary antibody PNA conjugate, or apre-deposited primary antibody recognized by a secondary antibody PNAconjugate). Of course, any of the PNA conjugates may have a PNA sequencehaving any number of nucleotides as described herein. Likewise, any ofthe PNA conjugates may have a PNA sequence including at least onenucleotide having a substituent at a gamma position, i.e. a gamma PNA.

In some embodiments, the sample may be contacted with two PNAconjugates, where each PNA conjugate is specific for a particulartarget, and where each PNA conjugate comprises a different PNA oligomerportion. In other embodiments, the sample may be contacted with threePNA conjugates, where each PNA conjugate is specific for a particulartarget, and where each PNA conjugate comprises a different PNA oligomerportion.

The PNA conjugates may be supplied to the tissue sample as a “pool” or“cocktail” comprising each of the PNA conjugates needed for theparticular assay. The pooling of PNA conjugates is believed to bepossible since the PNA conjugates are believed not to be cross-reactiveto each other, at least not to the extent where any cross-reactivitywould interfere with staining performance. Each PNA conjugate will bindto their respective targets and form detectable target-PNA conjugatecomplexes. In some embodiments, and following application of the PNAconjugates, a blocking step is performed.

Following the simultaneous application of the PNA conjugates (step 100),a plurality of detection reagents are simultaneously applied to thetissue sample (step 110), where each detection reagent facilitatesdetection of one of the PNA conjugates initially applied (at step 100),and where each detection reagent comprises a different reporter moiety.In some embodiments, the detection reagents are streptavidin conjugatedto a fluorophore, a chromophore, or a hapten. In other embodiments, thedetection reagents are secondary antibodies specific for a label of thePNA conjugate (e.g. anti-hapten antibodies specific to a hapten of thePNA conjugate). In yet other embodiments, the detection reagents are DNAor PNA sequences that are complementary to a PNA sequence of the PNAoligomer portion of the PNA conjugate. In embodiments where anti-labelantibodies are employed, the anti-label antibodies may be supplied tothe tissue sample as a pool or cocktail comprising each of theanti-label antibodies necessary for detection of the target-PNAconjugate complexes. Following application of the detection reagents, insome embodiments the tissue sample may be stained with a counterstain.Signals from each of the labels and/or reporter moieties may bevisualized or otherwise detected (e.g. simultaneously visualized ordetected).

One example of a multiplex assay utilizing PNA conjugates is as follows.A first PNA-antibody conjugate comprising a first PNA oligomer andspecific to a first target (e.g. specific to one of CD68, Ki67, CD20,etc.) is introduced to a tissue sample. In some embodiments, the firstPNA-antibody conjugate forms a detectable first target-PNA-antibodyconjugate complex. Simultaneously, a second PNA-antibody conjugatecomprising a second PNA oligomer and specific to a second target (e.g.another of CD68, Ki67, CD20, etc.) is introduced to the sample to form asecond target-PNA-antibody conjugate complex. Third, fourth, and nthadditional PNA-antibody conjugates to other targets (forming “n”target-detection probe complexes) and having different PNA sequencesand/or labels may be further introduced simultaneously with the firstand second PNA-antibody conjugates.

After the PNA-antibody conjugates are deposited, they may be detected,either directly or indirectly depending, of course, on theirconfiguration. In some embodiments, anti-label antibodies are introducedto enable detection of each of the target-PNA-antibody conjugatecomplex. In some embodiments, the anti-label antibodies are specific tothe different labels of the PNA conjugates, and where the anti-labelantibodies are each conjugated to a different reporter moiety. In someembodiments, the detectable reagents are anti-label antibodies eachconjugated to a fluorophore. In some embodiments, first, second, and nthanti-label antibodies are simultaneously introduced, where each of thefirst, second, and nth detection reagents are specific to the differentPNA-antibody conjugates, where each of the anti-label antibodies areconjugated to a fluorophore. In other embodiments, first, second, andnth anti-label antibodies are sequentially introduced, where each of thefirst, second, and nth detection reagents are specific to the differentPNA-antibody conjugates, and wherein each of the anti-label antibodiesare conjugated to an enzyme.

Alternatively, the PNA-antibody conjugates may be detected byintroducing PNA or DNA sequences that are complementary to PNA sequencesof the PNA oligomer portions of the introduced PNA conjugates. Eachcomplementary PNA or DNA sequence may comprise a reporter moiety asdetailed herein, including an enzyme, a fluorophore, a hapten, or ananoparticle. If the complementary PNA or DNA sequence comprises ahapten, additional detection reagents (e.g. anti-hapten antibodiesconjugated to an enzyme or fluorophore) may be introduced to facilitatedetection of the complementary PNA or DNA sequences, and hence thetargets within the sample.

As a further example of a multiplex assay according to the presentdisclosure, a first PNA-antibody conjugate specific to a first target(e.g. CD3, Ki67, PD-L1, or an immune cell marker) is introduced to atissue sample, the first PNA-antibody conjugate having a first label. Insome embodiments, the first PNA-antibody conjugate forms a detectablefirst target-PNA-antibody conjugate complex. Either simultaneously orsubsequently, a second PNA-antibody conjugate specific to a secondtarget (e.g. another of CD3, Ki67, PD-L1) is introduced to the sample toform a second target-PNA-antibody conjugate complex, the secondPNA-antibody conjugate having a second label. Third, fourth, and nthadditional PNA-antibody conjugates each specific to other targets(forming “n” target-PNA-antibody conjugate complexes) may be furtherintroduced, again either sequentially or simultaneously with the firstand/or second PNA-antibody conjugates, where the third, fourth and nthPNA-antibody conjugates each have yet different labels. After thePNA-antibody conjugates are deposited, they may be detected. In someembodiments, additional detection reagents are introduced to enable thedetection of the targets and the additional detection reagents includethose described herein (e.g. chromogenic detection reagents). In someembodiments, first, second, and nth detection reagents are sequentiallyintroduced, where each of the first, second, and nth detection reagentscomprise (i) a secondary antibody, namely an anti-label antibody,specific to each of the labels of the PNA-antibody conjugates, whereinthe secondary antibody is conjugated to an enzyme; and (ii) achromogenic substrate; wherein each of the first, second, and nthchromogenic substrates are different. In other embodiments, first,second, and nth detection reagents are sequentially introduced, whereeach of the first, second, and nth detection reagents comprise PNA orDNA sequences complementary to PNA sequences of the PNA oligomerportions of each of the PNA conjugates, each complementary PNA or DNAsequence comprising a reporter moiety.

As yet a further example of a multiplex assay according to the presentdisclosure, a first primary antibody specific to a first target (e.g.CD3, Ki67, PD-L1, or an immune cell marker) is introduced to a tissuesample (where the first primary antibody is not a conjugate of any ofFormulas (I), (IA), (IC), (ID), and (II)). Subsequently, a firstsecondary antibody-PNA conjugate specific to the first primary antibodyor a label conjugated to the first primary antibody is introduced, thefirst secondary-antibody PNA conjugate comprising a first PNA oligomerhaving a first PNA sequence. In some embodiments, the first secondaryantibody-PNA-antibody conjugate forms a detectable firsttarget-secondary antibody-PNA-antibody conjugate complex. Subsequently,the first PNA sequence of the secondary-antibody PNA conjugate iscleaved from the conjugate.

Subsequently, a second primary antibody specific to a second target(e.g. another of CD3, Ki67, PD-L1) is introduced to the sample.Subsequently, a second secondary antibody-PNA conjugate specific to thesecond primary antibody or a label conjugated to the second primaryantibody is introduced, the second secondary-antibody PNA conjugatecomprising a second PNA oligomer having a second PNA sequence. In someembodiments, the second secondary antibody-PNA-antibody conjugate formsa detectable second target-secondary antibody-PNA-antibody conjugatecomplex. Subsequently, the second PNA sequence of the secondary-antibodyPNA conjugate is cleaved from the conjugate. The skilled artisan willappreciate that any number of primary antibodies and secondary antibodyPNA-conjugates may be introduced sequentially, followed by cleavage ofthe PNA sequence from the PNA oligomer of the respective secondaryantibody PNA-conjugate. Finally, all of the different PNA sequences maybe measured, and the targets may be quantified.

In yet other embodiments, the multiplex detection method comprises thesteps of (i) contacting a biological sample with a first PNA-antibodyconjugate to form a first target antibody-PNA conjugate complex; (ii)contacting the biological sample with a first labeling conjugate whereinthe first labeling conjugate comprises a first enzyme (where the firstlabeling conjugate is an anti-label antibody that specifically binds tothe first PNA-antibody conjugate and is configured to label the targetwith an enzyme); (iii) contacting the biological sample with a firstsignaling conjugate comprising a first latent reactive moiety and afirst chromogenic moiety (see, e.g. U.S. patent application Ser. No.13/849,160, the disclosure of which is incorporated herein by referencefor a description of signaling conjugates and their constituentcomponents); (iv) inactivating the first enzyme, such as by contactingthe sample with a first enzyme inactivation composition to substantiallyinactivate or completely inactivate the first enzyme contained in thebiological sample.

After the first enzyme is inactivated (optional), the multiplex methodfurther comprises the steps of (v) contacting a biological sample with asecond PNA-antibody conjugate to form a second target-PNA-antibodyconjugate complex; (vi) contacting the biological sample with a secondlabeling conjugate wherein the second labeling conjugate comprises asecond enzyme (where the second labeling conjugate is an anti-labelantibody that specifically binds to the second PNA-antibody conjugateand is configured to label the target with an enzyme); (vii) contactingthe biological sample with a second signaling conjugate comprising asecond latent reactive moiety and a second chromogenic moiety; (viii)inactivating the second enzyme, such as by contacting the sample with afirst enzyme inactivation composition to substantially inactivate orcompletely inactivate the first enzyme contained in the biologicalsample.

After the second enzyme is inactivated, the method may be repeated suchthat additional PNA-antibody conjugates may be introduced, along withadditional detection reagents, to effectuate detection of other targets.Following introduction of all of the PNA-antibody conjugates (and otherdetection probes) and respective detection reagents or kits, the methodfurther comprises the step of counterstaining the sample and/ordetecting signals (manually or via an automated method) from the first,second, and nth chromogenic moieties, wherein each of the first, second,and nth chromogenic moieties are each different. Alternatively, each ofthe PNA-antibody conjugates may be added simultaneously or sequentially,but before any labeling conjugate is added. As another example, threePNA-antibody conjugates may be sequentially applied initially, prior tointroduction of any detection reagents, and then each of the detectionreagents added sequentially.

In the context of a multiplex assay where multiple targets are detectedsequentially, and where the detection employs the use of enzymes, it isdesirable to inactivate any reagent or endogenous enzymes betweensuccessive detection steps. As a result, it is believed that enzymespresent in any one detection step will not interfere with those in alater detection steps. This in turn is believed to improve upon thevisualization and detection of the different detectable moieties used inthe multiplex assay. Any enzyme inactivation composition known in theart may be used for this purpose. In some embodiments, an enzymeinactivation composition is applied to inactivate the reagent orendogenous enzymes after each detection step. Exemplary enzymeinactivation compositions are disclosed in co-pending application U.S.62/159,297, the disclosure of which is incorporated by reference hereinin its entirety.

In some embodiments, a denaturation step prevents the enzyme used in afirst set of detection reagents from acting on a second substrate. Insome embodiments, the denaturant is a substance that denatures theenzyme in the first detection reagent set. In some embodiments, thedenaturant is, for example, formamide, an alkyl-substituted amide, ureaor a urea-based denaturant, thiourea, guanidine hydrochloride, orderivatives thereof. Examples of alkyl-substituted amides include, butare not limited to, N-propylformamide, N-butylformamide,N-isobutylformamide, and N,N-dipropylaformamide. In some embodiments,the denaturant is provided in a buffer. For example, formamide may beprovided in a hybridization buffer comprising 20 mM dextran sulfate(50-57% % formamide (UltraPure formamide stock), 2×SSC (20×SSC stockcontaining 0.3 M citrate and 3M NaCl), 2.5 mM EDTA (0.5M EDTA stock), 5mM Tris, pH 7.4 (1 mM Tris, pH 7.4 stock), 0.05% Brij-35 (10% stockcontaining polyoxyethylene (23) lauryl ether), pH 7.4. In someembodiments, the sample is treated with the denaturant for a period oftime and under conditions sufficient to denature the first target probedetection enzyme, for example alkaline phosphatase. In some embodiments,the sample is treated with the denaturant for about 15 to about 30minutes, preferably about 20 to 24 minutes at about 37° C. In someembodiments, the sample is treated with the denaturant for a period oftime and under conditions sufficient to denature the target enzyme whilepreserving hybridization of the second nucleic acid probe to the target.

For those embodiments employing an anti-label antibody conjugated to anenzyme, conditions suitable for introducing the signaling conjugates orchromogenic substrates with the biological sample are used, andtypically include providing a reaction buffer or solution that comprisesa peroxide (e.g., hydrogen peroxide), and that has a salt concentrationand pH suitable for allowing or facilitating the enzyme to perform itsdesired function. In general, this step of the method is performed attemperatures ranging from about 35° C. to about 40° C., although theskilled artisan will be able to select appropriate temperature rangesappropriate for the enzymes and signalizing conjugates selected. Forexample, it is believed that these conditions allow the enzyme andperoxide to react and promote radical formation on the latent reactivemoiety of the signaling conjugate. The latent reactive moiety, andtherefore the signaling conjugate as a whole, will deposit covalently onthe biological sample, particularly at one or more tyrosine residuesproximal to the immobilized enzyme conjugate, tyrosine residues of theenzyme portion of the enzyme conjugate, and/or tyrosine residues of theantibody portion of the enzyme conjugate. The biological sample is thenilluminated with light and the target may be detected through absorbanceof the light produced by the chromogenic moiety of the signalingconjugate.

Methods of Detection with the Conjugates of any of Formulas (Ia), (Ib),(IIa), (IIb), and (IIc) in Conjunction with Other Specific BindingEntities

In some aspects of the present disclosure, the conjugates of any ofFormulas (IA), (IB), (IIA), (IIB), and (IIC) are used in conjugationwith other specific binding entities to effect multiplex detection oftargets in a tissue sample. The skilled artisan will appreciate that anyof the above-identified methods and procedures may be adaptedaccordingly for any assay employing both conjugates of any of Formulas(IA), (IB), (IIA), (IIB), and (IIC) and other specific binding entities.

In some embodiments, the other specific binding entities include nucleicacids for in situ hybridization and unmodified antibodies for IHC. Asused herein, the terms “unmodified antibody” or “unmodified antibodies”refer to those antibodies that do not comprise a nucleotide sequence(e.g. a DNA, PNA nucleotide sequence, or gamma PNA nucleotide sequenceas identified herein), but includes those antibodies conjugated to ahapten or another label. In essence, “unmodified antibodies” are nativeantibodies traditionally used in IHC assays, which are specific to aparticular target (e.g. an anti-CD3 antibody) and which may be detected,such as with anti-species secondary antibodies or, if they comprise alabel, an anti-label antibody. By way of example, a rabbit anti-CD3antibody may be detected with a goat anti-rabbit antibody. Likewise, arabbit anti-CD3 antibody conjugated to a hapten may be detected with ananti-hapten antibody.

FIGS. 14B and 14C illustrate methods for the multiplex detection oftargets where a tissue sample is contacted with one or more unmodifiedprimary antibodies (simultaneously or sequentially) (first stage, 220)and then subsequently contacted with one or more PNA conjugates(simultaneously or sequentially) (second stage, 250). The skilledartisan will recognize that the first stage 220 and the second stage 250may be reversed, such that the PNA conjugates are applied first to thetissue sample followed by application of the unmodified antibodies. Theskilled artisan will also appreciate that appropriate nucleic acidprobes (including those conjugated to a label) may be substituted forthe unmodified antibodies such that the multiplex assay includes bothISH and IHC steps or stages (in any order).

In some embodiments, such as depicted in FIG. 14B, a first unmodifiedprimary antibody may be applied to a tissue sample to form a firsttarget-primary antibody complex (step 200). Next, first detectionreagents specific to the unmodified primary antibody are applied to thetissue sample to detect the first target-primary antibody complex (step210). Dotted line 205 in FIG. 14B illustrates that steps 200 and 210 offirst stage 220 may be repeated one or more times to provide for thesequential multiplex detection of multiple, different targets within thetissue sample with unmodified primary antibodies. For example, a secondunmodified primary antibody be applied to the tissue sample to form asecond target-primary antibody complex (200), followed by application ofsecond detection reagents specific to the second unmodified primaryantibody detect the second target-primary antibody complex (210).

FIG. 14C represents an alternative method for the multiplex detection oftargets using a two-stage method similar to that presented in FIG. 14B.In the method depicted in FIG. 14C, each of the unmodified antibodyconjugates are simultaneously introduced to the tissue sample at step260. Next, the sample is contacted with detection reagents (e.g.anti-species antibodies or anti-label antibodies) at step 270 toeffectuate detection of the unmodified antibodies. In an alternativeembodiment, all of the unmodified primary antibodies may be sequentiallyapplied (step 260), followed by sequential application of the respectiveanti-species antibodies (step 270) (or, where appropriate, anti-haptenantibodies).

The skilled artisan will appreciate that the detection reagents for theunmodified antibodies may comprise anti-species antibodies specific tothe utilized unmodified antibodies. Alternatively, the detectionreagents for the unmodified antibodies may comprise anti-haptenantibodies specific to haptens conjugated to the unmodified antibodies.The skilled artisan will also appreciate that the anti-species oranti-hapten antibodies may comprise a reporter moiety and, inembodiments where the reporter moiety is an enzyme, additionalchromogenic substrates may be supplied with the first and seconddetection reagents.

Following the first stage of the multiplex assay 220 (FIG. 14B or 14C),a second stage 250 is performed, where the tissue sample issimultaneously or sequentially contacted with a plurality of PNAconjugates (step 230), where each PNA conjugate is specific for aparticular target, and where each PNA conjugate comprises a differentPNA oligomer portion. The PNA conjugates may be supplied to the tissuesample as a “pool” or “cocktail” comprising each of the PNA conjugatesneeded for the particular assay. Each PNA conjugate will form adetectable target-PNA conjugate complex with a specific target.Following the simultaneous or sequential application of the PNAconjugates (step 230), anti-label antibodies (secondary antibodies) aresimultaneously applied to the tissue sample (step 240), where eachanti-label antibody is specific to one of PNA conjugates applied, andwhere each anti-label antibody comprises a different reporter moiety.The anti-label antibodies may be supplied to the tissue sample as a“pool” or “cocktail” comprising each of the anti-label antibodiesnecessary for detection of the target-PNA-antibody complexes.

Alternatively, following introduction of the respective PNA conjugatesat step 230, the sample may be contacted at step 240 with a PNA sequenceor a DNA sequence that is complementary to the PNA sequence of the PNAconjugate, the DNA sequence comprising a reporter moiety (e.g. anenzyme, chromogen, a fluorophore, or a hapten). In embodiments where theDNA sequence comprises a chromogen or a fluorophore, the “labeled”target complex may be directly detected. On the other hand, where theDNA sequence comprises a hapten, an anti-hapten antibody conjugated to areporter moiety must be introduced to facilitate eventual detection ofthe “labeled” target complex.

Of course, and as an alternative embodiment, PNA sequences of therespective PNA conjugates may be quantified such as with the NanoStringnCounter method described herein.

Following step 250, in some embodiments the tissue sample may be stainedwith a counterstain. Signals from each of the reporter moieties (e.g.from the anti-species or anti-label antibodies) may be visualized orotherwise detected (e.g. simultaneously visualized or detected).

As an example of a multiplex assay comprising both (i) unmodifiedantibodies, and (ii) PNA-antibody conjugates according to the presentdisclosure, a first antibody conjugate comprising a hapten label (e.g.an anti-CD3 antibody conjugated indirectly to a happen) is introduced toa tissue sample to form a target-antibody-conjugate complex.Simultaneously, an unmodified antibody (e.g. a rabbit anti-PDL1antibody) is introduced to the tissue sample to form atarget-unmodified-antibody complex. Next, detection reagents areintroduced (simultaneously or sequentially) to detect the formedtarget-antibody-conjugate complex (e.g. an anti-happen antibody) and theformed target-unmodified-antibody complex (e.g. a goat anti-rabbitantibody), where each of the detection reagents are conjugated to adifferent fluorophore.

In a second stage of the multiplex assay, a first PNA-antibody conjugatecomprising a first PNA oligomer and specific to a first target (e.g.specific to CD68) is introduced to a tissue sample. In some embodiments,the first PNA-antibody conjugate forms a detectable firsttarget-PNA-antibody conjugate complex. Sequentially or simultaneously, asecond PNA-antibody conjugate comprising a second PNA oligomer andspecific to a second target (e.g. specific to Ki67) is introduced to thesample to form a second target-PNA-antibody conjugate complex. Third,fourth, and nth additional PNA-antibody conjugates specific to othertargets (forming “n” target-PNA-antibody complexes) and having differentPNA oligomers may be further introduced simultaneously with the firstand second PNA-antibody conjugates. In alternative embodiments,PNA-antibody conjugates may be added sequentially, wherein the PNAsequence is cleaved from a PNA-antibody conjugate prior to introductionof a subsequent PNA-antibody conjugate. The cleaved PNA sequences maythen be measured together.

After the PNA-antibody conjugates are deposited, they may be detected,either directly or indirectly depending, of course, on theirconfiguration. In some embodiments, first, second, and nth detectionreagents are simultaneously introduced, where each of the first, second,and nth detection reagents are specific to the different PNA-antibodyconjugates. In other embodiments, first, second, and nth detectionreagents are sequentially introduced, where each of the first, second,and nth detection reagents are specific to the different PNA-antibodyconjugates. In some embodiments, anti-label antibodies are introduced toenable detection of each of the target-PNA-antibody conjugatescomplexes. In some embodiments, the detection reagents are anti-labelantibodies that are specific to the different labels of the PNA-antibodyconjugates and where the anti-label antibodies are each conjugated to areporter moiety, e.g. a fluorophore or an enzyme. In some embodiments,the detectable reagents are anti-label antibodies each conjugated to afluorophore. In other embodiments, the detectable reagents areanti-label antibodies each conjugated to an enzyme. In yet otherembodiments, the detectable reagents are a combination of anti-labelantibodies conjugated to a fluorophore and anti-label antibodiesconjugated to an enzyme. In those embodiments where the anti-labelantibodies are conjugated to an enzyme, substrates for the enzymes areprovided to effect detection (as noted previously herein). In otherembodiments, PNA or DNA sequences complementary to one or more of PNAsequences and comprising enzymes, fluorophores or haptens are introducedto enable detection of each of the target-PNA-antibody conjugatescomplexes. In yet other embodiments, the detection reagents are PNA orDNA sequences that are complementary to a PNA sequence of the PNAoligomer portion of the PNA conjugate. The skilled artisan willappreciate that where complementary PNA or DNA sequences are providedwhich are conjugated to a hapten, further reagents may be supplied tothe sample to facilitate detection of the complementary PNA or DNAsequences. In alternative embodiments, the PNA-conjugated antibodies maybe hybridized to a complementary PNA or DNA sequence ex situ and thenthe complex, i.e. PNA-conjugated antibody hybridized to thecomplementary PNA or DNA sequence) may be introduced to the tissue toenable labeling of targets within the sample.

Automation

The multiplex assays and methods may be automated and may be combinedwith a specimen processing apparatus. The specimen processing apparatuscan be an automated apparatus, such as the BENCHMARK XT instrument andSYMPHONY instrument sold by Ventana Medical Systems, Inc. VentanaMedical Systems, Inc. is the assignee of a number of United Statespatents disclosing systems and methods for performing automatedanalyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809,6,352,861, 6,827,901 and 6,943,029, and U.S. Published PatentApplication Nos. 2003/0211630 and 2004/0052685, each of which isincorporated herein by reference in its entirety. Alternatively,specimens can be manually processed.

The specimen processing apparatus can apply fixatives to the specimen.Fixatives can include cross-linking agents (such as aldehydes, e.g.,formaldehyde, paraformaldehyde, and glutaraldehyde, as well asnon-aldehyde cross-linking agents), oxidizing agents (e.g., metallicions and complexes, such as osmium tetroxide and chromic acid),protein-denaturing agents (e.g., acetic acid, methanol, and ethanol),fixatives of unknown mechanism (e.g., mercuric chloride, acetone, andpicric acid), combination reagents (e.g., Carnoy's fixative, methacarn,Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid),microwaves, and miscellaneous fixatives (e.g., excluded volume fixationand vapor fixation).

If the specimen is a sample embedded in paraffin, the sample can bedeparaffinized with the specimen processing apparatus using appropriatedeparaffinizing fluid(s). After the waste remover removes thedeparaffinizing fluid(s), any number of substances can be successivelyapplied to the specimen. The substances can be for pretreatment (e.g.,protein-crosslinking, expose nucleic acids, etc.), denaturation,hybridization, washing (e.g., stringency wash), detection (e.g., link avisual or marker molecule to a probe), amplifying (e.g., amplifyingproteins, genes, etc.), counterstaining, coverslipping, or the like.

The specimen processing apparatus can apply a wide range of substancesto the specimen. The substances include, without limitation, stains,probes, reagents, rinses, and/or conditioners. The substances can befluids (e.g., gases, liquids, or gas/liquid mixtures), or the like. Thefluids can be solvents (e.g., polar solvents, non-polar solvents, etc.),solutions (e.g., aqueous solutions or other types of solutions), or thelike. Reagents can include, without limitation, stains, wetting agents,antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.),antigen recovering fluids (e.g., aqueous- or non-aqueous-based antigenretrieval solutions, antigen recovering buffers, etc.), or the like.Probes can be an isolated nucleic acid or an isolated syntheticoligonucleotide, attached to a detectable label or reporter molecule.Labels can include radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

After the specimens are processed, a user can transport specimen-bearingslides to the imaging apparatus. The imaging apparatus used here is abrightfield imager slide scanner. One brightfield imager is the iScanCoreo™ brightfield scanner sold by Ventana Medical Systems, Inc. Inautomated embodiments, the imaging apparatus is a digital pathologydevice as disclosed in International Patent Application No.:PCT/US2010/002772 (Patent Publication No.: WO/2011/049608) entitledIMAGING SYSTEM AND TECHNIQUES or disclosed in U.S. Patent PublicationNo. 2014/0178169 filed on Sep. 9, 2011, entitled IMAGING SYSTEMS,CASSETTES, AND METHODS OF USING THE SAME.

Counterstaining Counterstaining is a method of post-treating the samplesafter they have already been stained with agents to detect one or moretargets, such that their structures can be more readily visualized undera microscope. For example, a counterstain is optionally used prior tocoverslipping to render the immunohistochemical stain more distinct.Counterstains differ in color from a primary stain. Numerouscounterstains are well known, such as hematoxylin, eosin, methyl green,methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI(4′,6-diamidino-2-phenylindole) is a fluorescent stain that may be used.

In some examples, more than one stain can be mixed together to producethe counterstain. This provides flexibility and the ability to choosestains. For example, a first stain, can be selected for the mixture thathas a particular attribute, but yet does not have a different desiredattribute. A second stain can be added to the mixture that displays themissing desired attribute. For example, toluidine blue, DAPI, andpontamine sky blue can be mixed together to form a counterstain.

Imaging

Certain aspects, or all, of the disclosed embodiments can be automated,and facilitated by computer analysis and/or image analysis system. Insome applications, precise color or fluorescence ratios are measured. Insome embodiments, light microscopy is utilized for image analysis.Certain disclosed embodiments involve acquiring digital images. This canbe done by coupling a digital camera to a microscope. Digital imagesobtained of stained samples are analyzed using image analysis software.Color or fluorescence can be measured in several different ways. Forexample, color can be measured as red, blue, and green values; hue,saturation, and intensity values; and/or by measuring a specificwavelength or range of wavelengths using a spectral imaging camera. Thesamples also can be evaluated qualitatively and semi-quantitatively.Qualitative assessment includes assessing the staining intensity,identifying the positively-staining cells and the intracellularcompartments involved in staining, and evaluating the overall sample orslide quality. Separate evaluations are performed on the test samplesand this analysis can include a comparison to known average values todetermine if the samples represent an abnormal state.

Samples and Targets

Samples include biological components and generally are suspected ofincluding one or more target molecules of interest. Target molecules canbe on the surface of cells and the cells can be in a suspension, or in atissue section. Target molecules can also be intracellular and detectedupon cell lysis or penetration of the cell by a probe. One of ordinaryskill in the art will appreciate that the method of detecting targetmolecules in a sample will vary depending upon the type of sample andprobe being used. Methods of collecting and preparing samples are knownin the art.

Samples for use in the embodiments of the method and with thecomposition disclosed herein, such as a tissue or other biologicalsample, can be prepared using any method known in the art by of one ofordinary skill. The samples can be obtained from a subject for routinescreening or from a subject that is suspected of having a disorder, suchas a genetic abnormality, infection, or a neoplasia. The describedembodiments of the disclosed method can also be applied to samples thatdo not have genetic abnormalities, diseases, disorders, etc., referredto as “normal” samples. Such normal samples are useful, among otherthings, as controls for comparison to other samples. The samples can beanalyzed for many different purposes. For example, the samples can beused in a scientific study or for the diagnosis of a suspected malady,or as prognostic indicators for treatment success, survival, etc.

Samples can include multiple targets that can be specifically bound by aprobe or reporter molecule. The targets can be nucleic acid sequences orproteins. In some examples, the target is a protein or nucleic acidmolecule from a pathogen, such as a virus, bacteria, or intracellularparasite, such as from a viral genome. For example, a target protein maybe produced from a target nucleic acid sequence associated with (e.g.,correlated with, causally implicated in, etc.) a disease.

The skilled artisan will appreciate that PNA conjugates may be developedwhich are specific to any of the following targets:

In specific, non-limiting examples, a target protein is produced by atarget nucleic acid sequence (e.g., genomic target nucleic acidsequence) associated with a neoplasm (for example, a cancer). Numerouschromosome abnormalities (including translocations and otherrearrangements, amplification or deletion) have been identified inneoplastic cells, especially in cancer cells, such as B cell and T cellleukemias, lymphomas, breast cancer, colon cancer, neurological cancersand the like. Therefore, in some examples, at least a portion of thetarget molecule is produced by a nucleic acid sequence (e.g., genomictarget nucleic acid sequence) amplified or deleted in at least a subsetof cells in a sample.

In other examples, a target protein produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) that is a tumorsuppressor gene that is deleted (lost) in malignant cells. For example,the p16 region (including D9S1749, D9S1747, p16(INK4A), p14(ARF),D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21 is deletedin certain bladder cancers. Chromosomal deletions involving the distalregion of the short arm of chromosome 1 (that encompasses, for example,SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), and thepericentromeric region (e.g., 19p13-19q13) of chromosome 19 (thatencompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, andGLTSCR1) are characteristic molecular features of certain types of solidtumors of the central nervous system.

Numerous other cytogenetic abnormalities that correlate with neoplastictransformation and/or growth are known to those of ordinary skill in theart. Target proteins that are produced by nucleic acid sequences (e.g.,genomic target nucleic acid sequences), which have been correlated withneoplastic transformation and which are useful in the disclosed methods,also include the EGFR gene (7p12; e.g., GENBANK™ Accession No.NC-000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21;e.g., GENBANK™ Accession No. NC-000008, nucleotides128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene(8p22; e.g., GENBANK™ Accession No. NC-000008, nucleotides19841058-19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC-000013,nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK™ AccessionNo. NC-000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24;e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No.NC-000012, complement, nucleotides 56196638-56200567), FUS (16p11.2;e.g., GENBANK™ Accession No. NC-000016, nucleotides 31098954-31110601),FKHR (13p14; e.g., GENBANK™ Accession No. NC-000013, complement,nucleotides 40027817-40138734), as well as, for example: ALK (2p23;e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK™Accession No. NC-000011, nucleotides 69165054.69178423), BCL2 (18q21.3;e.g., GENBANK™ Accession No. NC-000018, complement, nucleotides58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC-000003,complement, nucleotides 188921859-188946169), MALF1, API (1p32-p31;e.g., GENBANK™ Accession No. NC-000001, complement, nucleotides59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No.NC-000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3;e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No.NC-000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3;e.g., GENBANK™ Accession No. NC-000007, complement, nucleotides13897379-13995289), EWS (22q12.2; e.g., GENBANK™ Accession No.NC-000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g.,GENBANK™ Accession No. NC-000011, nucleotides 128069199-128187521), PAX3(2q35-q37; e.g., GENBANK™ Accession No. NC-000002, complement,nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™Accession No. NC-000001, nucleotides 18830087-18935219), PTEN (10q23.3;e.g., GENBANK™ Accession No. NC-000010, nucleotides 89613175-89716382),AKT2 (19q13.1-q13.2; e.g., GENBANK™ Accession No. NC-000019, complement,nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ AccessionNo. NC-000001, complement, nucleotides 40133685-40140274), REL(2p113-p12; e.g., GENBANK™ Accession No. NC-000002, nucleotides60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK™ Accession No.NC-000005, complement, nucleotides 149413051-149473128).

EXAMPLES

The non-limiting examples presented herein each incorporate the use ofPNA conjugates. Applicants submit that the PNA conjugates disclosedherein are suitable for use in IHC assays. Of course, as detailedherein, the PNA conjugates may be used in conjunction with otherdetectable specific binding entities and may be utilized in assays whichcombine IHC and ISH.

Example 1: PNA/Antibody Conjugation

Exemplary conjugation of PNA to goat-anti-mouse (GAM), goat-anti-rabbit(GAR), and mouse-anti-DIG, is described below:

(1) 100 micrograms of antibody (0.667 nmole) was diluted in PBS to 2mg/mL and treated with 15 molar equivalent of SPDP-PEG-NHS (10 nmolefrom 10 mM DMSO stock solution) for 2 hours. SPDP-PEG-NHS was availablefrom Quanta Biodesign, Ohio, USA (SPDP-dPEG8-NHS ester, product #10376).

(2) The labeled antibody was purified with 7 KD MW cutoff Zeba spindesalting columns from ThermoFisher (product #89882).

(3) 2 nmole PNA (8 μL from 251 μM PNA stock solution dissolved 1:1 (v/v)water DMF), was added to the antibody solution with the addition of 5 μLDMF, and 35 μL of PBS to yield approximately total of 100 μL of reactionsolution. The mixture was incubated overnight at room temperature.

(4) The conjugates were then purified using Zeba spin desalting columnswere then used to purify PNA-conjugated antibodies from the reactionmixture.

The purified PNA-conjugated antibodies were diluted in a suitablediluent before use.

Example 2: UV-Vis Measurement of Antibody-PNA Conjugates

The PNA-conjugated antibodies were characterized with UV-Vis absorbance.The increase of absorbance at 260 nm in the antibody-PNA conjugatesindicated successful incorporation of PNA (see FIGS. 2A and 2B). Theratio of A260 to A280 nm may be used to qualitatively assess theefficiency of conjugation and potentially estimate the number of PNAoligomers per antibody. This is also illustrated in FIGS. 30 and 31 .

Example 3: Detection of PNA-Antibody Conjugate Using SA-HRP

To prove that the conjugation of the PNA oligomers (15 bases PNA, 10bases PNA or 15 bases gamma PNA) does not affect the binding affinityand specificity of the antibody, we performed IHC assays on tonsiltissues for different markers using PNA-conjugated antibodies (primaryand secondary). All IHC assays were run on Benchmark XT (Ventana) usingprotocols that were modified based on the specific assay. ThePNA-conjugated antibodies were manually added as a titration at a volumeof 100 μL and at appropriate concentrations.

The biotin label on the PNA tag was detected. Two tonsil slides wereincubated with anti-CD45 and anti-Ki67 primary antibodies followed byincubation with PNA-conjugated GAR and GAM secondary antibodies (PNAsequence having 15 bases), respectively. The slides were then incubatedwith Streptavidin-HRP (SA-HRP) followed by DAB deposition (chromogenicdetection). The markers were observed at their expected locations (FIGS.3A and B). No background signal was observed when primary antibodieswere omitted from the assays (FIG. 3C). Tonsil slides were alsosuccessfully stained against CK5/6 using primary haptenated antibodies(Anti CK5/6:DIG) and secondary PNA-conjugated anti-hapten antibodiesfollowed by SA-HRP and DAB deposition (FIGS. 4A and 4B). The sameexperiment was performed with a PNA sequence having 10 bases. Tonsiltissue was stained with anti-Ki67 then GAR conjugated to the PNAsequence have 10 bases. The biotin moiety at the end of the PNA sequencehaving 10 bases conjugated to the GAR was detected using SA-HRP followedby DAB deposition (FIG. 21A). As a comparison tonsil tissue was stainedfor Ki67 with GAR-HRP (conventional Ultraview detection by Ventana). Theexperiment showed that the 10 base PNA sequence conjugation did notalter the functionality of GAR.

Tonsil was stained with anti-Ki67 then GAR conjugated to a gamma PNAsequence (15 bases). The biotin at the end of the gamma PNA sequence wasdetected through a SA-HRP followed by DAB deposition (FIG. 28 ). Thefigure shows that gamma PNA can be successfully conjugated to secondaryantibody without causing any mis-localization.

These first experiment provides strong evidence that different PNAsequence lengths (10 bases and 15 bases) and nature (conventional andgamma PNA) can be efficiently conjugated to secondary antibodies and theconjugation does not affect their binding affinity and specificity tothe corresponding primary antibodies on tonsil slides.

PNA tags (10 bases PNA and 15 bases gamma PNA) were directly conjugatedto primary antibodies and detected through a biotin moiety on the PNAsequence as previously described. Tonsil slides were incubated withanti-CD3, anti-CD8, anti-CD34, anti-Ki67 primary antibodies conjugatedto a PNA sequence having 10 bases that has a biotin on its end (referredto as sPNA1). The slides were then incubated with Streptavidin-HRP(SA-HRP) followed by DAB deposition (chromogenic detection). All markerswere observed at their expected locations. FIGS. 22A, 22B, 22C, 22D,shows the tonsil slides stained with anti-CD3 conjugated to 10 basesPNA, anti-CD8 conjugated to 10 bases PNA, anti-CD34 conjugated to 10bases PNA, and anti-Ki67 conjugated to 10 bases PNA respectively

FIGS. 23 and 24 shows tonsil slides stained with anti-CD3 and anti-CD8respectively conjugated to a different 10 bases PNA sequence referred toas sPNA2 that has a biotin on its end and detected as previouslydescribed.

Tonsil slides were also stained with antiCD3, anti-CD8 and antiPD-L1conjugated to 15 bases gamma PNA sequence. The biotin moiety on thegamma PNA sequence was detected through incubation with SA-HRP followedby DAB deposition. FIGS. 29 A, B and C show tonsil slides stained withanti-CD3, anti-CD8 and anti-PD L1 at different concentrations,respectively.

Example 4A: Chemical Cleavage of the PNA Oligo

In some embodiments, the PNA-conjugate antibody is designed to allowmultiplexed quantitative measurement of protein expression by methodssuch as Gyros technology or the NanoString nCounter platform, which isbased on DNA counting. Therefore, the PNA sequence must be cleaved fromthe PNA-conjugated antibody after binding to the tissue to allow ex situPNA counting. In this particular example, the PNA sequence was bound tothe antibody via a disulfide bond, which could be chemically cleaved(e.g. by reduction of the disulfide bond) leading to PNA sequencerelease.

FIGS. 5A to 5D shows tonsil slides stained for Ki67 and CD45 withprimary antibodies followed by PNA-conjugated anti-species secondaryantibodies and detected with SA-HRP DAB deposition. Incubating theslides with 20 mM TCEP (Tris(2-carboxyethyl)phosphine, reducing agent)before SA-HRP treatment resulted in the total loss of the brown stainingcolor, which indicated the removal of the PNA tags.

Example 4B: Photo-Cleavage of the PNA Oligo

Tonsil slides were treated with primary antibody (rabbit Ki67),secondary antibody (GAR-PL-PNA, goat anti rabbit antibody withphotocleavable PNA that has biotin on it). Slides were then irradiatedwith UV light (hand-held UV lamp, 365 nm) for different time periods. Acontrol slide was not treated with UV and used for comparison. Allslides were then treated with SA-HRP and DAB for detection.

FIGS. 12A, 12B, and 12C illustrate that the irradiated slides do nothave color, indicating the photocleavage of the PNA sequence. In thisexperiment, the whole slides were irradiated with UV light. The cleavageof PNA from the whole slides can be achieved with chemical reduction ofthe disulfide bond. Light irradiation gives the possibility ofselectively irradiating specific area of interest on the slide. To provethis concept, we have used a Laser Capture Microdissection (LCM) systemto achieve selective irradiation. The LCM used UV light that camethrough an objective (high spatial precision) to cut specific area ofthe tissue (down to single cell). We have used UV to selectivelyirradiate specific areas on the tissue and photocleave the PNA. FIG. 13illustrates tonsil slides where a germinal center was irradiated (−nostaining) next to another germinal center that was not irradiated(positive staining). The LCM UV is 355 nm.

Example 5: Fluorescent Detection of the PNA/Antibody Conjugate UsingSA-FITC

Besides chromogenic detection, biotin in the conjugate can also befluorescently using SA-fluorophore. FIGS. 6A and 6B show a fluorescentimage of a tonsil slide stained for Ki67 with a primary antibodyfollowed by PNA-conjugated secondary antibody and detected with SA-FITC.SA-FITC binds biotin on the PNA sequence (see FIG. 6C). The fluorescentsignal is consistent with the localization of the Ki67 marker. Thisexperiment demonstrated the versatility of detection schemes thePNA-conjugated antibody provides.

Example 6: Quantitative Measurement of PNA

Without wishing to be bound by any particular theory, it has beenreported that the NanoString nCounter platform has the ability topotentially detect as high as 800 different DNA sequences allowingtremendous multiplexing capability for IHC once the antibody PNAconjugate is used. We assume that PNA can be detected in a similarmanner to DNA as the detection scheme is based on the hybridization of areporter strand to the target oligo (can presumably could be DNA orPNA). It is believed that the PNA oligomers can be significantly shorterthan the standard DNA targets typically used with the NanoStringnCounter platform (which are typically about 100 bases in length)because the presence of the biotin on the 3′ end of the PNA excludes theneed to use a capture strand. Moreover, the higher binding affinity ofPNA to DNA compared to DNA to DNA is believed to provide enoughstability to the relatively short PNA/DNA reporter duplex.

As a proof of concept, we showed that the fluorescently stained slidedescribed in Example 8 herein could be incubated with TCEP (20 mM) toremove the PNA-SA-FITC. Released PNA-SA-FITC was measured based on thefluorescence intensity. The number of PNA-conjugated antibodies bound tothe slide could then be estimated (FIGS. 7A and 7B)

Example 7: Detection of PNA Using Complement Fluorescent DNA

In the previous experiments the PNA was detected by staining against thebiotin label. Complement DNA or PNA sequences carrying different labels(chromogenic, fluorogenic, etc.) may be used as alternative stainingapproaches. In this experiment, tonsil slides were incubated with rabbitanti-Ki67 primary antibody followed by PNA-conjugated GAR. Then, theslide was incubated with a DNA sequence complement to the PNA havingfluorescent labels (e.g. FITC or Rhodamine). FIGS. 8A, 8B, 8C, and 8Dshow that the slides were successfully stained through DNAhybridization. The fluorescent stains were consistent with the markerlocalization and no background signal was observed. DNA sequences wereadded as a manual titration at a concentration of about 185 nM andvolume of about 100 μL.

DNA sequences:

1: (SEQ ID NO: 16) 5′-CTGAAGATGGTTGAC/Rhodamine-3′ 2: (SEQ ID NO: 17)5′-FAM/CTGAAGATGGTTGAC-3′

This experiment demonstrates that PNA tag conjugated to the antibody isactive and accessible through hybridization with complementary DNA ontissue. The two DNA sequences were complements to the same PNA, witheach carrying a unique fluorophore. The two fluorophores were placed ondifferent ends of the DNA sequence (FITC on the 5′ end and Rhodamine onthe 3′ end). Consequently, FITC was located at the far end away from theantibody after hybridization with PNA, whereas Rhodamine was placed inthe close proximity of the antibody. Positive staining was observed inboth cases, proving the capability of detecting the PNA withhybridization to the complementary DNA sequences.

In another experiment, tonsil tissue was stained with anti-CD3conjugated with 10 bases PNA sequence 1 and anti-CD8 conjugated with 10bases PNA sequence 2 (PNA sequence 1 is different than PNA sequence 2).The two PNA-conjugated antibodies were added simultaneously as acocktail (mixture). Then two 10 bases DNA sequences complementary to the2 PNA sequences such that DNA sequence 1 is complementary to PNAsequence 1 and has an alexa488 fluorophore on its end and DNA sequence 2is complimentary to PNA sequence 2 and has alexa647 fluorophore on itsend. The two DNA sequences were added as a cocktail (mixture).Fluorescence images show that when we look under the green channel, allCD3 positive cells were shown (FIG. 27A) and when we look under the redchannel, all CD8 positive cells were shown (FIG. 27B). The merged imageshows the co-localization of the two markers (FIG. 27C).

Example 8: Detection of PNA Using Complement Haptenated DNA

In this experiment, the complement DNA was labeled with a hapten (DIG).Two tonsil slides were incubated with rabbit anti-Ki67 and mouseanti-CD45 followed by PNA-conjugated GAR and PNA-conjugated GAMrespectively. DIG-labeled complement DNA was incubated with both slidesfollowed by HRP conjugated anti-DIG antibody and DAB deposition. DNA wasadded as a manual titration step at a concentration of about 185 nM anda volume of about 100 μL and temperature of 37° C.

FIGS. 9A, 9B, and 9C show that slides were successfully stained and thestaining pattern was consistent with the localization of the markers. Nobackground signal was observed when primary antibodies were omitted.

DNA sequence: (SEQ ID NO: 18) 5′-CTGAAGATGGTTGAC/DIG/-3′

FIG. 25 illustrates a similar experiment performed with shorter PNAsequence (10 bases). Tonsil tissue was stained with anti-Ki67 then GARconjugated to short PNA (10 bases) followed by incubation with 10 basesDNA sequence that is complementary to the 10 bases PNA tag. DNA sequenceis labeled with a DIG use to visualize the staining with Anti-DIG-HRPantibody followed by DAB deposition.

Example 9: PNA Ab Conjugation Via Click Chemistry

The steps of conjugating a PNA oligomer to an antibody are as follows:

(1) Antibody reduction to introduce sulfhydryl group (thiols): to 100 μgof Ab add 2.5 μL of 1 M DTT (Dithiothreitol) and incubate for 30 min

(2) Remove excess DTT with Zeba desalting spin column (7 MWCO)

(3) Add DBCO-maleimide heterobifunctional linker (from click chemistrytools A108-25) at the ratio 1:12 Ab:linker and incubate overnight

(4) Clean with Zeba column and add azide-PNA with ration 1:6Ab:azide-PNA and incubate overnight.

(5) Clean with Zeba column.

FIG. 19 illustrates tonsil tissue incubated with anti-Ki67 primaryantibody (Rabbit mAb) and GAR-PNA (conjugated with click chemistry) anddetected with SA-HRP. The image shows that the PNA was successfullyconjugated via click chemistry to the Antibody.

Example 10: PNA Ab Conjugation Via Maleimide Chemistry

The steps of conjugating a PNA oligomer to an antibody are as follows:

(1) Antibody reduction to introduce sulfhydryl group (thiol): to 100 μgof Ab add 2.5 μL of 1 M DTT (Dithiothreitol) and incubate for 30 min;

(2) Remove excess DTT with Zeba desalting spin column (7 MWCO);

(3) Add maleimide-PNA the ratio 1:6 Ab:maleimide DNA and incubateovernight; and

(4) Clean with Zeba column

Example 11: PNA Quantification Using the Gyros Platform Technology

To demonstrated quantification of PNA using Gyros technology, abiotinylated PNA oligo (Bt-PNA) with concentration ranging from about0.0274 nM to about 20 nM was tested (Table 1). For each concentration, a1:2 ratio of Bt-PNA to a complementary single stranded DNA labeled withdigoxigenin (DNA-DIG) was first hybridized at room temperature beforebeing analyzed by Gyros in duplicates. The detection was achieved usingMs-anti-DIG followed by Alexa Fluor 647-GAM. A four-point curve (seeTable 1) was fitted using the Gyros program, and a standard curve withR² greater than 0.998 was generated. Notably, the signal to backgroundratio (S/B) of the lowest PNA concentration was about 10 about 10,suggesting very low background signal due to PNA or ssDNA.

TABLE 1 Standard DruveCurve of PNA Having a Concentration ranging from0.0274 nM to 20 nM Exp Conc Calu Conc Ave Conc, CV Conc Bias Sample ID[nM] Response S/B [nM] [nM] [%] [%] Blank 0 0.0576 1.07 Blank 0 0.05030.933 Bt-PNA 1 0.0274 0.509 9.44 0.0263 0.0277 6.83 −3.94 Bt-PNA 10.0274 0.559 10.4 0.029 0.0277 6.83 5.81 Bt-PNA 2 0.0823 1.43 26.60.0813 0.0804 1.51 −1.27 Bt-PNA 2 0.0823 1.41 26.1 0.0795 0.0804 1.51−3.36 Bt-PNA 3 0.247 3.52 65.3 0.229 0.25 12 −7.41 Bt-PNA 3 0.247 4.0775.5 0.271 0.25 12 9.73 Bt-PNA 4 0.74 9.68 180 0.762 0.785 4.16 2.97Bt-PNA 4 0.74 10.2 188 0.808 0.785 4.16 9.21 Bt-PNA 5 2.22 22.9 424 2.22.16 2.9 −0.749 Bt-PNA 5 2.22 22.1 410 2.11 2.16 2.9 −4.74 Bt-PNA 6 6.6752 965 6.55 6.58 0.632 −1.86 Bt-PNA 6 6.67 52.4 971 6.6 6.58 0.632−0.975 Bt-PNA 7 20 105 1940 19.1 20.3 8.55 −4.44 Bt-PNA 7 20 112 208021.6 20.3 8.55 7.86

To further demonstrate that PNA cleaved from the antibody conjugate canbe quantified and the antibody can be further stained after PNAcleavage, experiments were performed as shown in FIG. 33 . Threeantibodies (Ki67, CD8 and PD-L1) were conjugated with Bt-PNA-1 and usedfor detecting the respective marker in normal tonsil tissue section.Most of the tissue processing steps were performed using a VentanaBenchMark XT autostainer. After standard antigen retrieval, PNA labeledprimary antibodies were applied to tissue and incubated at about 37° C.for about 16 minutes. The slides were then removed from the tissuestainer and rinsed with reaction buffer and water. To each slide about100 μL of 20 mM TCEP solution was added and incubated in a humidity boxfor about 20 minutes. Eighty microliters of the solution containing thecleaved PNA was collected and further quantified by Gyros (such as byusing the techniques described herein). The slides after PNA cleavagewere placed back in the autostainer and the primary antibody was furtherdetected using a Ventana UltraView DAB universal detection kit.

The quantification of the cleaved Bt-PNA was achieved throughhybridization with excess of complementary single strandedDNA-digoxigenin following the same procedure as previously described.The results were summarized in Table 2. The results clearly suggest thatPNA from antibody conjugates for tissue staining can be successfullycleaved and further quantified using Gyros technology.

TABLE 2 Quantification of PNA Cleaved from Tissue Exp Cal Conc Conc BiasSample ID [pM] Response S/B [pM] [%] Standards Blank 0 0.748 1 Standard1 5000 117 156 5020 0.394 Standard 2 1000 36.2 48.4 984 −1.56 Standard 3200 10.3 13.7 205 2.58 Standard 4 40 2.74 3.66 39.4 −1.6 Standard 5 80.897 1.2 8.03 0.425 Samples Ki67 A⁽¹⁾ unknown 12.2 16.3 255 N/A Ki67B⁽¹⁾ unknown 14.4 19.2 310 N/A CD8 A⁽¹⁾ unknown 1.85 2.47 23.3 N/A CD8B⁽¹⁾ unknown 2.39 3.19 32.9 N/A PD-L1 A⁽¹⁾ unknown 4.62 6.18 76.6 N/APD-L1 B⁽¹⁾ unknown 4.94 6.6 83.2 N/A Negative Bt-PNA 125 nM⁽²⁾ 1.01 1.359.67 N/A Controls Bt-PNA 12.5 nM⁽²⁾ 0.529 0.707 <8.00 N/A DIG-DNA 250nM⁽³⁾ 0.553 0.739 <8.00 N/A TCEP solution 0.398 0.532 <8.00 N/A⁽¹⁾DIG-DNA final concentration 250 nM ⁽²⁾NO DIG-DNA added, PNA only asnegative control ⁽³⁾NO Bt-PNA added, DIG-DNA only as negative control

After PNA cleavage, the antibody on the very same tissue section canstill be stained by standard IHC for visualization of the PNA encodedmarker as shown in FIGS. 34A and 34B. Specific staining of each markerwas observed, suggesting the cleavage conditions did not causenoticeable damage to the tissue or the binding of antibody to themarker. This result suggests a new way to combine quantification withvisualization of spatial distribution of biomarkers in FFPE tissue,which is a unique advantage of the antibody-PNA conjugates.

Example 12—Comparison of Different PNA Oligomers Detected by GyrosTechnology

The procedure set forth in Example 11 was utilized. The following PNAoligomers and DNA oligomers were compared:

PNA 1: Biotin-o-GTCAACCATCTTCAG-Lys(C6SH)-3′(where GTCAACCATCTTCAG comprises SEQ ID NO: 1) sPNA1:Biotin-o-CCATCTTCAG-Lys(SMCC) (where CCATCTTCAG comprises SEQ ID NO: 4)Gamma PNA (gPNA): (SEQ ID NO: 15)Biotin-O-GT*-CAA*-CCA*-TCT*-TCA*-G-Lys(SMCC) Short Gamma PNA (sgPNA):(SEQ ID NO: 19) Biotin-O-CCA*-TCT*-TCA*-G-Lys(SMCC) DNA: Biotin-GTCAACCATCTTCAG (where GTCAACCATCTTCAG comprises SEQ ID NO: 1)cDNA-DIG for detection: (SEQ ID NO: 18) 5′-DIG-CTGAAGATGG-3′

Final Concentrations of PNA tested: 250 μM, 50 μM, 10 μM and 2 μM

Final Concentration of cDNA-DIG for detection: 25 nM

The PNA or DNA oligomer was incubated with cDNA-DIG at room temperaturebefore being analyzed by Gyros. The results of the analysis areillustrated in FIGS. 35A and 35B.

As shown in FIG. 35A, all PNA oligomers were able to be detected at thelowest concentration (2 μM), but the DNA oligomers were not able to bedetected at the same concentration (low S/B ratio). For allconcentrations, the response from PNA oligomers were much higher thanwith the DNA oligomers. This demonstrated the superior stability of thePNA-DNA hybrid. The response difference among different PNA's was notsignificant.

FIG. 35B illustrates that the signal-to-background (S/B) ratio of thePNA oligomers was much higher than the corresponding DNA oligomers,especially at lower concentrations (lower panel). Overall, the resultssuggested that PNA oligomers (even the 10-mer sequences) could bequantified to low-pM concentrations using Gyros. On the other hand, DNAoligomers (15-mer) having the same sequence could not be quantified tosuch low-pM concentrations. In addition, gamma-PNA oligomers afforded ahigher S/B ratio relative to the corresponding PNA oligomers.

Example 13A

In this experiment, tonsil tissue was stained with Ki67 (rabbit Ab), andthen a Goat Anti-Rabbit (GAR) antibody conjugated to PNA sequence(CCATCTTCAG). A DNA sequence that was complementary to the PNA oligomerand having a DIG reporter moiety on its end was incubated with theGAR-PNA on the slide. After washing, an anti-DIG:HRP Ab was added.Subsequently, DAB was deposited to generate a brown signal. The presenceof the signal would indicate that the DNA successfully hybridized to thePNA since the signal requires the presence of DIG on the DNA. Theabsence of the signal (as shown in FIG. 36A) indicated that at theexperimental conditions (200 nM of DNA, 37° C., 30 min) the DNA did nothybridize. It is believed that the DNA PNA affinity is not strong enoughto assure hybridization with such a short PNA sequence at theexperimental conditions.

Example 13B

In this experiment, the PNA from Example 13A is replaced with a gammaPNA (15 bases, but only 10 bases are complementary to the same DNAsequence). The same experimental conditions from Example 13A wereapplied. As compared with Example 12, the presence of the signalindicates that gammaPNA/DNA duplex is much more stable than PNA/DNA ofthe same hybridizing sequence and at the same condition (see FIG. 36B).Indeed, the presence of the signal indicated that the gamma DNAhybridizes to the gamma PNA to generate the brown signal. Since theexperimental conditions are the same as in Example 13A, the experimentdemonstrated that the gamma PNA DNA affinity was higher than the PNADNA, which shows the superiority of the gamma PNA compared to a shortPNA as in Example 13A.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

Although the disclosure herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent disclosure. It is therefore understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present disclosure as defined by the appended claims.

Additional Embodiments

-   -   Additional Embodiment 1. A PNA conjugate having the structure of        Formula (IIB):

-   -   -   wherein        -   ‘Specific binding entity’ is selected from the group            consisting of an antibody, an antibody fragment, a            drug/antibody complex, and a nucleic acid;        -   ‘Linker’ is a branched or unbranched, linear or cyclic,            substituted or unsubstituted, saturated or unsaturated,            group having between 2 and 80 carbon atoms, and optionally            having one or more heteroatoms selected from O, N, or S;        -   ‘PNA’ is a PNA sequence having at least one nucleotide            including a substitution at a gamma carbon position;        -   X is selected from the group consisting of biotin, an            enzyme, a chromogen, a fluorophore, a hapten, and a mass            spectrometry tag;        -   Y is a branched or unbranched, linear or cyclic, substituted            or unsubstituted, saturated or unsaturated group having            between 1 and 12 carbon atoms and optionally having one or            more O, N, or S heteroatoms;        -   m is 0 or an integer ranging from 1 to 6;        -   z is 0 or 1; and        -   n is an integer ranging from 1 to 12.

    -   Additional Embodiment 2. The PNA conjugate of additional        embodiment 1, wherein ‘Specific binding entity’ is an antibody.

    -   Additional Embodiment 3. The PNA conjugate of additional        embodiment 1 or 2, wherein the at least one substituted        nucleotide is selected from the group consisting of a lysine        residue, a peptide sequence having less than 20 amino acids, a        polymer, and a miniPEG.

    -   Additional Embodiment 4. The PNA conjugate of additional        embodiment 3, wherein at least one reporter moiety is coupled to        the at least one substituted nucleotide.

    -   Additional Embodiment 5. The PNA conjugate of any of the        preceding additional embodiments, wherein m is 0, z is 0, and n        is greater than 1.

    -   Additional Embodiment 6. The PNA conjugate of additional        embodiment 5, wherein n is an integer ranging from between 2 and        6.

    -   Additional Embodiment 7. The PNA conjugate of any of the        preceding additional embodiments, wherein ‘Linker’ comprises at        least one hydrophilic group.

    -   Additional Embodiment 8. The PNA conjugate of any of the        preceding additional embodiments, wherein ‘Linker’ has the        structure depicted in Formula (IVA):

-   -   -   wherein        -   d and e are integers each independently ranging from 2 to            20;        -   Q is a bond, O, S, or N(R^(c))(R^(d));        -   R^(a) and R^(b) are independently H, a C₁-C₄ alkyl group, F,            Cl, or N(R^(c))(R^(d));        -   R^(c) and R^(d) are independently CH₃ or H; and        -   A and B are independently a branched or unbranched, linear            or cyclic, substituted or unsubstituted, saturated or            unsaturated group having between 1 and 12 carbon atoms and            optionally having one or more O, N, or S heteroatoms.

    -   Additional Embodiment 9. The PNA conjugate of additional        embodiment 8, wherein d and e are integers ranging from 2 to 6.

    -   Additional Embodiment 10. The PNA conjugate of additional        embodiment 8, wherein at least one of A or B includes a        cleavable moiety.

    -   Additional Embodiment 11. The PNA conjugate of additional        embodiment 10, wherein the cleavable moiety is a photocleavable        group.

    -   Additional Embodiment 12. The PNA conjugate of additional        embodiment 10, wherein the cleavable moiety is a chemically        cleavable group.

    -   Additional Embodiment 13. The PNA conjugate of any of the        preceding additional embodiments, wherein ‘Specific binding        entity’ is an antibody, ‘Linker’ comprises at least one alkylene        oxide group, m is 0, z is 0, and n is greater than 1.

    -   Additional Embodiment 14. The PNA conjugate of any of the        preceding additional embodiments, wherein ‘Specific binding        entity’ is an antibody, ‘Linker’ comprises at least one alkylene        oxide group, and n is greater than 1.

    -   Additional Embodiment 15. The PNA conjugate of additional        embodiment 14, wherein ‘Linker’ further comprises at least one        cleavable moiety.

    -   Additional Embodiment 16. The PNA conjugate of additional        embodiment 15, wherein X is a hapten.

    -   Additional Embodiment 17. A PNA oligomer having the structure of        Formula (III):

-   -   -   wherein        -   T is a group having between 1 and 4 carbon atoms and which            is optionally substituted with O, N, or S and having a            terminal reactive moiety;        -   ‘Linker’ is a branched or unbranched, linear or cyclic,            substituted or unsubstituted, saturated or unsaturated,            group having between 2 and 80 carbon atoms, and optionally            having one or more heteroatoms selected from O, N, or S;        -   ‘PNA’ is a PNA sequence having at least one nucleotide            including a substitution at a gamma carbon position;        -   X is selected from the group consisting of biotin, an            enzyme, a chromogen, a fluorophore, a hapten, and a mass            spectrometry tag;        -   Y is a branched or unbranched, linear or cyclic, substituted            or unsubstituted, saturated or unsaturated group having            between 1 and 12 carbon atoms and optionally having one or            more O, N, or S heteroatoms;        -   m is 0 or an integer ranging from 1 to 6; and        -   z is 0 or 1.

    -   Additional Embodiment 18. The PNA oligomer of additional        embodiment 17, wherein the at least one substituted nucleotide        is selected from the group consisting of a lysine residue, a        peptide sequence having less than 20 amino acids, a polymer, and        a miniPEG.

    -   Additional Embodiment 19. The PNA conjugate of additional        embodiment 17 or 18, wherein at least one reporter moiety is        coupled to the at least one substituted nucleotide.

    -   Additional Embodiment 20. The PNA oligomer of any of additional        embodiments 17 to 19, wherein m is 0, z is 0, and n is greater        than 1.

    -   Additional Embodiment 21. The PNA oligomer of any of additional        embodiments 17 to 20, wherein n is an integer ranging from        between 2 and 6.

    -   Additional Embodiment 22. The PNA oligomer of any of additional        embodiments 17 to 21, wherein ‘Linker’ has the structure        depicted in Formula (IVA):

-   -   -   wherein        -   d and e are integers each independently ranging from 2 to            20;        -   Q is a bond, O, S, or N(R^(c))(R^(d));        -   R^(a) and R^(b) are independently H, a C₁-C₄ alkyl group, F,            Cl, or N(R^(c))(R^(d));        -   R^(c) and R^(d) are independently CH₃ or H; and        -   A and B are independently a branched or unbranched, linear            or cyclic, substituted or unsubstituted, saturated or            unsaturated group having between 1 and 12 carbon atoms and            optionally having one or more O, N, or S heteroatoms.

    -   Additional Embodiment 23. The PNA oligomer of additional        embodiment 22, wherein d and e are integers ranging from 2 to 6.

    -   Additional Embodiment 24. The PNA oligomer of additional        embodiment 22, wherein at least one of A or B comprises a        cleavable moiety.

    -   Additional Embodiment 25. The PNA oligomer of additional        embodiment 24, wherein the cleavable moiety is a photocleavable        group.

    -   Additional Embodiment 26. The PNA oligomer of additional        embodiment 24, wherein the cleavable moiety is a chemically        cleavable group.

    -   Additional Embodiment 27. A PNA conjugate comprising the PNA        oligomer of any of additional embodiments 17 to 26 and a        specific binding entity.

    -   Additional Embodiment 28. The PNA conjugate of additional        embodiment 27, wherein the specific binding entity is a primary        antibody; and wherein the PNA sequence or gamma PNA sequence        comprises between 5 and 30 bases.

    -   Additional Embodiment 29. A method of synthesizing a PNA        conjugate comprising reacting the PNA oligomer of any of        additional embodiments 17 to 26 with a specific binding entity.

    -   Additional Embodiment 30. A method of detecting a target in a        sample, comprising:

contacting the sample with a first PNA conjugate, the first PNAconjugate having the structure of Formula (IIB):

-   -   wherein    -   ‘Specific binding entity’ is selected from the group consisting        of an antibody, an antibody fragment, a drug/antibody complex,        and a nucleic acid;    -   ‘Linker’ is a branched or unbranched, linear or cyclic,        substituted or unsubstituted, saturated or unsaturated, group        having between 2 and 80 carbon atoms, and optionally having one        or more heteroatoms selected from O, N, or S;    -   ‘PNA’ is a PNA sequence having at least one nucleotide including        a substitution at a gamma carbon position;    -   X is selected from the group consisting of biotin, an enzyme, a        chromogen, a fluorophore, a hapten, and a mass spectrometry tag;    -   Y is a branched or unbranched, linear or cyclic, substituted or        unsubstituted, saturated or unsaturated group having between 1        and 12 carbon atoms and optionally having one or more O, N, or S        heteroatoms;    -   m is 0 or an integer ranging from 1 to 6;    -   z is 0 or 1; and    -   n is an integer ranging from 1 to 12; and

contacting the sample with first detection reagents to facilitatedetection of the PNA conjugate.

-   -   Additional Embodiment 31. The method of additional embodiment        30, wherein the ‘Specific binding entity’ is a primary antibody        and wherein the primary antibody is specific to a first target.    -   Additional Embodiment 32. The method of additional embodiment 30        or 31, wherein the ‘Specific binding entity’ is a secondary        antibody, and wherein the method further comprises the step of        contacting the sample with a primary antibody specific for a        first target prior to contacting the sample with the first PNA        conjugate, and wherein the first PNA conjugate is specific to        the first primary antibody.    -   Additional Embodiment 33. The method of any of additional        embodiments 30 to 32, wherein the first detection reagents are        anti-label antibodies specific to a label of the PNA conjugate.    -   Additional Embodiment 34. The method of any of additional        embodiments 30 to 33, wherein the label is a hapten and the        anti-label antibodies are anti-hapten antibodies.    -   Additional Embodiment 35. The method of any of additional        embodiments 30 to 34, wherein the detection reagents comprise a        PNA or DNA sequence complementary to a PNA sequence or a gamma        PNA sequence of the first PNA conjugate, the complementary PNA        or DNA sequence conjugated to a reporter moiety.    -   Additional Embodiment 36. The method of additional embodiment        35, wherein the reporter moiety is a fluorophore.    -   Additional Embodiment 37. The method of additional embodiment        35, wherein the reporter moiety is a hapten, and where the        method further comprises contacting the sample with anti-hapten        antibodies specific to the hapten of the complementary PNA or        DNA sequence.    -   Additional Embodiment 38. A method of detecting a target in a        sample, comprising:

contacting the sample with a first PNA conjugate, the first PNAconjugate having the structure of Formula (IIB):

-   -   wherein    -   ‘Specific binding entity’ is selected from the group consisting        of an antibody, an antibody fragment, a drug/antibody complex,        and a nucleic acid;    -   ‘Linker’ is a branched or unbranched, linear or cyclic,        substituted or unsubstituted, saturated or unsaturated, group        having between 2 and 80 carbon atoms, and optionally having one        or more heteroatoms selected from O, N, or S, wherein the        ‘Linker’ includes a cleavable moiety;    -   ‘PNA’ is a PNA sequence having at least one nucleotide including        a substitution at a gamma carbon position;    -   X is selected from the group consisting of biotin, an enzyme, a        chromogen, a fluorophore, a hapten, and a mass spectrometry tag;    -   Y is a branched or unbranched, linear or cyclic, substituted or        unsubstituted, saturated or unsaturated group having between 1        and 12 carbon atoms and optionally having one or more O, N, or S        heteroatoms;    -   m is 0;    -   z is 0; and    -   n is an integer ranging from 1 to 12; and contacting the sample        with a reagent to cleave the cleavable moiety on the ‘Linker,’        and

quantifying an amount of the cleaved ‘PNA’ sequence.

-   -   Additional Embodiment 39. The method of additional embodiment        38, wherein the quantification of the amount of the PNA sequence        is performed using NanoString nCounter technology, Gyros        technology, or mass spectrometry.    -   Additional Embodiment 40. The method of additional embodiment 38        or 39, wherein the cleavable group is selected from the group        consisting of a photocleavable group, a chemically cleavable        group, or an enzymatically cleavable group.    -   Additional Embodiment 41. The method of any of additional        embodiments 38 to 40, further comprising visualizing the        Specific Binding Entity from which the PNA sequence was cleaved.    -   Additional Embodiment 42. A conjugate of Formula (IB):

-   -   -   wherein        -   ‘Specific binding entity’ is selected from the group            consisting of an antibody, an antibody fragment, a            drug/antibody complex, and a nucleic acid;        -   ‘Linker’ is a branched or unbranched, linear or cyclic,            substituted or unsubstituted, saturated or unsaturated,            group having between 2 and 80 carbon atoms, and optionally            having one or more heteroatoms selected from O, N, or S;        -   Z is a PNA sequence having at least one nucleotide including            a substitution at a gamma carbon position;        -   X is selected from the group consisting of biotin, an            enzyme, a chromogen, a fluorophore, a hapten, and a mass            spectrometry tag;        -   Y is a branched or unbranched, linear or cyclic, substituted            or unsubstituted, saturated or unsaturated group having            between 1 and 12 carbon atoms and optionally having one or            more O, N, or S heteroatoms;        -   m is 0 or an integer ranging from 1 to 6;        -   z is 0 or 1; and        -   n is an integer ranging from 1 to 12.

    -   Additional Embodiment 43. The conjugate of additional embodiment        42, wherein the ‘Specific Binding Entity’ is a primary antibody.

    -   Additional Embodiment 44. The conjugate of additional embodiment        42 or 43, wherein the PNA sequence has between about 5 and about        30 bases.

    -   Additional Embodiment 45. The conjugate of any of additional        embodiments 42 to 44, wherein the PNA sequence has about 10        bases.

    -   Additional Embodiment 46. The conjugate of any of additional        embodiments 42 to 45, wherein the at least one substituted        nucleotide is selected from the group consisting of a lysine        residue, a peptide sequence having less than 20 amino acids, a        polymer, and a miniPEG.

    -   Additional Embodiment 47. The conjugate of additional embodiment        46, wherein at least one reporter moiety is coupled to the at        least one substituted nucleotide.

The invention claimed is:
 1. A PNA oligomer having the structure ofFormula (III):

wherein T is a group having between 1 and 4 carbon atoms and which isoptionally substituted with O, N, or S and having a terminal reactivemoiety; ‘Linker’ is a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated, group havingbetween 2 and 80 carbon atoms, and optionally having one or moreheteroatoms selected from O, N, or S; ‘PNA’ is a PNA sequence having atleast one nucleotide including a substitution at a gamma carbonposition; X is selected from the group consisting of biotin, an enzyme,a chromogen, a fluorophore, a hapten, and a mass spectrometry tag; Y isa branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms; mis an integer ranging from 1 to 6; and z is 0 or
 1. 2. The PNA oligomerof claim 1, wherein the substitution in the gamma carbon position in thePNA nucleotide is selected from the group consisting of a lysineresidue, a peptide having less than 20 amino acids, a polymer, and aminiPEG.
 3. The PNA conjugate of claim 1, wherein at least one reportermoiety is coupled to the at least one substituted nucleotide.
 4. The PNAoligomer of claim 1, wherein m is 0, and z is
 0. 5. The PNA oligomer ofclaim 1, wherein ‘Linker’ included a moiety having Formula (IVA):

wherein d and e are integers each independently ranging from 2 to 20; Qis a bond, O, S, or N(R^(c))(R^(d)); R^(a) and R^(b) are independentlyH, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d)); R^(c) and R^(d) areindependently CH₃ or H; and A and B are independently a branched orunbranched, linear or cyclic, substituted or unsubstituted, saturated orunsaturated group having between 1 and 12 carbon atoms and optionallyhaving one or more O, N, or S heteroatoms.
 6. The PNA oligomer of claim5, wherein d and e are integers ranging from 2 to
 6. 7. The PNA oligomerof claim 5, wherein at least one of A or B comprises a cleavable moiety.8. The PNA oligomer of claim 7, wherein the cleavable moiety is aphotocleavable group.
 9. The PNA oligomer of claim 7, wherein thecleavable moiety is a chemically cleavable group.
 10. The PNA oligomerof claim 1, wherein the ‘Linker’ group comprises at least onehydrophilic group.
 11. The PNA oligomer of claim 1, wherein thesubstitution at the gamma carbon position comprises a miniPEG.
 12. ThePNA oligomer of claim 1, wherein the substitution at the gamma carbonposition comprises a polymer.
 13. The PNA oligomer of claim 1, whereinthe substitution at the gamma carbon position comprises an oligoethylenemoiety.
 14. The PNA oligomer of claim 1, wherein T comprises a clickadduct.
 15. A method of synthesizing a PNA conjugate comprising reactingthe PNA oligomer of claim 1 with a specific binding entity, wherein thespecific binding entity is an antibody, and where the PNA oligomer ofclaim 1 is reacted with one of an amine, thiol, or carbohydrate residueof the antibody.
 16. A PNA conjugate having the structure of Formula(IIB):

wherein ‘Specific binding entity’ is selected from the group consistingof an antibody, an antibody fragment, a drug/antibody complex, and anucleic acid; ‘Linker’ is a branched or unbranched, linear or cyclic,substituted or unsubstituted, saturated or unsaturated, group havingbetween 2 and 80 carbon atoms, and optionally having one or moreheteroatoms selected from O, N, or S; ‘PNA’ is a PNA sequence having atleast one nucleotide including a substitution at a gamma carbonposition; X is selected from the group consisting of biotin, an enzyme,a chromogen, a fluorophore, a hapten, and a mass spectrometry tag; Y isa branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms; mis an integer ranging from 1 to 6; z is 0 or 1; and n is an integerranging from 1 to
 12. 17. The PNA conjugate of claim 16, wherein‘Specific binding entity’ is an antibody.
 18. The PNA conjugate of claim16, wherein the at least one substituted nucleotide is selected from thegroup consisting of a lysine residue, a peptide sequence having lessthan 20 amino acids, a polymer, and a miniPEG.
 19. The PNA conjugate ofclaim 18, wherein at least one reporter moiety is coupled to the atleast one substituted nucleotide.
 20. A PNA oligomer having thestructure of Formula (III):

wherein T is selected from the group consisting of NHS ester, thiol,maleimide, and azide; ‘Linker’ is a branched or unbranched, linear orcyclic, substituted or unsubstituted, saturated or unsaturated, grouphaving between 2 and 80 carbon atoms, and optionally having one or moreheteroatoms selected from O, N, or S; ‘PNA’ is a PNA sequence having atleast one nucleotide including a substitution at a gamma carbonposition; X is selected from the group consisting of biotin, an enzyme,a chromogen, a fluorophore, a hapten, and a mass spectrometry tag; Y isa branched or unbranched, linear or cyclic, substituted orunsubstituted, saturated or unsaturated group having between 1 and 12carbon atoms and optionally having one or more O, N, or S heteroatoms; mis 0 or an integer ranging from 1 to 6; and z is 0 or 1.